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Waters 2489 UV/Visible Detector Operator`s Guide

1. 3 28 Preparing the Detector Press DIAG and then press 2 Sample amp ref energy The sample and reference energy diagnostic display appears Sample and reference energy diagnostic display AP GOED SOMple 2 i 1231227 ina Ref 230 Reis l 243 250 ind Record the numbers for later comparison Rule Run this procedure each time you change the detector lamp Flush the flow cell with approximately 30 to 60 mL of HPLC grade methanol at 1 mL min for 15 minutes minimum to clean the cell Make the plumbing connections described in Making plumbing connections on page 2 6 before beginning the peak response test Verifying peak response This test evaluates the detector s peak response To run the test 1 2 3 4 5 6 On the absorbance screen use the arrow keys to highlight the A field Enter 254 into the A field and then press Enter Press Enter again to activate the sensitivity field Set the sensitivity to 2 0 AUFS Set the pump flow rate in your HPLC system to 1 0 mL min Inject 1 uL acetone When the test is successful your chart recorder or data system displays a peak Using the operator interface 3 29 Wavelength calibration You can calibrate the detector manually from the keypad at any time during operation or if calibration arise errors during startup You need not reboot the detector after a successful wavelength calibration To calibrate the detector manua
2. Zero scan screen 4 of 4 Sample scan screen 3 of 3 Scanning spectra Running the sample scan Run the zero scan before running the sample scan To ensure identical flow cell and solvent conditions run the sample scan for the corresponding zero scan within 15 minutes of running the zero scan To run a sample scan 1 Set up the zero or reference scan by following the steps in the zero scan procedure on page 3 47 On the first New scan screen press 1 Sample Scan Result The parameters for wavelength range AUFS Pace and Mark tick mark you entered for the corresponding zero scan appear Press Next to advance to the second sample scan screen Tip You can change the entries in the AUFS and Mark each fields Press Next to advance to the third sample scan screen and then press Run Result A brief message Initializing appears The Scanning screen shows the progress of the scan in nanometers across a progress bar Scanning progress bar Scanning O S 0 299 nm 0 6356 AU After a brief pause the detector displays a sample scan graphically Sample erbium scan graphical display DEBE 379 r m T bal m 1 2 T 3 50 Preparing the Detector Tip To return to the Scan choice list at the end of the scan press SCAN Shift Chart mark Press Next Result Doing so displays as many as four of the highest peaks scanned within the specified ra
3. Fix voltage Contact closures amp events Previous choices 4 Lamp display amp keypad gt gt List of diagnostic tests used to test lamp display and keypad functions Change lamp Test keypad Test display Previous choices 5 Service Diagnostic tests used by Waters Service personnel 6 Other diagnostics gt gt Diagnostic tests that generate test peaks with which you determine wavelength accuracy or override the default filter setting Generate test peaks Optical filter override Previous choices Using the diagnostic tests The detector employs both user selectable and service diagnostic tests You access user diagnostic tests by pressing the DIAG key Only qualified Waters service personnel can access service diagnostic tests Using the sample and reference energy diagnostic tests You use the sample and reference energy diagnostic tests to plot the output of the analog channels to examine noise fluctuations and to compare with the User selected diagnostic tests 5 11 AU time trace The current sample and reference energy readings appear in nanoamps nA Sample and reference energy diagnostic test To use the sample and reference energy diagnostic test 1 2 3 Press DIAG and then press 2 Change the wavelength if necessary Press Enter Result When the new wavelength shifts to the left the corresponding sa
4. Index 12 filter 4 5 general considerations C 2 C 3 guidelines C 2 miscibility C 3 C 6 reservoirs C 6 UV cutoff C 9 C 11 viscosity considerations C 6 spare parts 4 3 sparging C 8 specifications bandwidth B 2 dimensions B 4 drift B 2 electrical B 3 environmental B 3 filter setting B 2 flow cell 4 15 B 5 linearity B 2 operating B 1 B 5 optical B 4 optical component B 3 repeatability B 2 sensitivity setting B 2 wavelength accuracy B 2 wavelength range B 2 spectra generating 3 9 new 3 46 3 53 obtaining information about 3 54 replaying 3 56 reviewing 3 55 scanning 3 42 3 60 scanning using a static flow cell 3 59 storing 3 53 subtracting 3 55 spectral features 1 9 spectrum scanning 1 12 stand alone operation 3 26 starting detector 3 2 methods from the separations module 2 15 run 3 23 run clock 3 9 wavelength 3 42 startup diagnostic tests 3 2 diagnostic tests failure 3 3 errors 3 30 5 2 kit 2 24 2 26 sticky diagnostic tests 3 6 5 9 stopping the run clock 3 9 stored spectra obtaining information 3 54 reviewing information 3 55 storing methods 3 34 3 39 spectra 3 53 subtracting a spectrum 3 55 switch 1 timed event parameter 3 35 switch 2 timed event parameter 3 36 switched outputs 2 10 switches programming 3 24 setting 5 15 symbols caution A 5 electrical A 12 handling A 13 warning A 2 system displaying information 3 11 information 3 25 specifications B 1 B 5 System Info key 3 11
5. Instrument error messages Continued Error Message Description Corrective Action System not calibrated The calibration read 1 Cycle power to the from nonvolatile detector memory is not valid 2 Perform manual calibration 3 Contact Waters Technical Service User selected diagnostic tests Overview You can run several diagnostic tests for troubleshooting the detector and verifying that its electronics and optics perform correctly To perform user selectable diagnostic tests 1 Press the DIAG key from the detector s front panel Diagnostic tests choice list Reset instrument z Sample amp ref energy 3 nput amp autput gt 4Lamp display amp keypad gt 5 Other diagnostics gt ad Zeck 2 To access a specific diagnostic test press the up or down arrow key to select a test and then press Enter Alternative Press a number on the keypad corresponding to the test number Choices that display other choices are indicated by gt gt Sticky diagnostic tests remain in effect until you disable them When a test is active the detector absorbance screen displays a wrench icon see the figure below You can disable a specific sticky diagnostic test by resetting it to the default settings User selected diagnostic tests 5 9 You can disable all active sticky diagnostic tests by pressing DIAG and then selecting 1 Reset instrument If no sticky diagnostic te
6. display the hours and minutes the lamp has been turned on during the current run and or since installation 3 60 Preparing the Detector To turn off the lamp manually from the detector front panel 1 Press Lamp Shift 1 from the keypad The lamp control screen appears Lamp control screen Press QT again r7 to turn lamp cU Lamp has been on Oh OF mi Times Total s ines installed Fired 12h 03 r 5 2 Press Lamp Shift 1 again to turn the lamp off Lamp off on sequence Lamp off indicator Lamp on indicator AP Igniting gt AES AES Ass 0 00 2 0000 aurs 0 00 0 00 2 0000 ar pe 20000 AUFS pes To ignite the lamp manually 1 When there is an X through the lamp icon on the absorbance screen press Lamp Shift 1 2 Press Lamp Shift 1 again to turn the lamp on The absorbance screen appears with the message Igniting Result The lamp can take as many as 1 minute to illuminate Once the lamp illuminates the display returns to the absorbance screen and the X is removed from the lamp icon You can conserve lamp life by programatically igniting and extinguishing it for example overnight using a timed event method To ignite or extinguish the lamp programatically select the Timed events option in the Method choice list or program the lamp through one of the external contact closures Scanning spectra 3 61 See Programming timed events threshold events and methods on page
7. 11 Press Next again to return to the sample scan display 12 When you finish manipulating the sample scan graphical display press SCAN Shift Chart mark to return to the Scan choice list To store the scan see Storing a spectrum on page 3 53 The figure below shows a series of scans of anthracene dissolved in acetonitrile to display the use of the Scale function The zero scan is not displayed For the scaling parameters AU1 and AU2 the default is Auto You can change the AU parameter based on the absorbance of the spectrum To return the default to auto press CE 3 52 Preparing the Detector Series of scans of anthracene in acetonitrile Sample scan 200 nm to 400 nm 0 001 AU to 0 5 AU anthracene Zoom of sample scan 200 nm to 300 nm 0 001 AU to 0 5 AU anthracene 230 nm to 270 nm A2 changed to 300 nm Zoom of sample scan 230 nm to 270 nm 0 001 AU to 0 5 AU anthracene 250 nm 41 changed to 230 nm A2 changed to 270 nm AU1 and AU2 on auto Zoom of sample scan 35 375 300 nm to 400 nm 0 001 AU to 0 025 AU anthracene 330 nm to 400 nm 41 changed to 300 nm 42 changed to 400 nm AU1 and AU2 on auto Storing a spectrum Once you run a spectrum you can store it for later review subtraction or playback You can store as many as three spectra Scanning spectra 3 53 To store a spectrum 1 3 4 From the graphic display of a sample scan return to the first Scan screen by pre
8. 2 For the outputs SW1 and SW2 a Press Enter to display the active switch surrounded by a dotted line border b Press any numerical key to change the status of the switch ON to OFF or vice versa c Press Enter to select the second switch Using the lamp display and keypad diagnostic tests To access the lamp display and keypad diagnostic tests press DIAG and then press 4 User selected diagnostic tests 5 15 Using the change lamp diagnostic test Caution Make sure the detector is powered off and the power cord is disconnected before you replace the lamp To change the lamp 1 From the Lamp display amp keypad choice list press 1 Change lamp to access the Change Lamp diagnostic display Change Lamp diagnostic display Serial number of lamp 7 1235456759 Install date Year Hours e Bfes o7 e 2 Enter the 9 digit serial number and date of installation for the new lamp Press Enter after each entry Tip Use this function whenever you change the lamp to enter a new serial number and installation date see page 4 21 and page 4 23 fora complete explanation of the lamp replacement procedure Caution Neglecting to record the new lamp serial number following the prescribed procedures referenced above voids the lamp warranty The date of the previous lamp installation remains in the detector memory 5 16 Error Messages Diagnostic Tests and Troubleshooting To use the Test keypad d
9. Initializing the detector entsteet ege 3 2 IS nn me P 3 3 Using the Operator interlace siccsivsssissessssesessccessssvsessievesssavsesseusecsesdesessesessessesss 3 4 eae he disp i ritchie idee 3 4 Liuns ebd vH eai e ID EM uM ce rr mer eee 3 7 Navigating the user interface iue esi e xe a e HERR EVE prx ERE R QE 3 12 Primary and secondary EE 3 14 Operating other detector Dame vsissicensecciveiavaesseryeisasessasaesarseseareceaaeenns 3 22 Operating the detector cessisse nennen nnne nnne nennen enne 3 26 Verifying that the detector operates proper 3 27 We CG AT OP ea tov isin iia br Pl PO tise eee 3 30 Operating the detector in single wavelength mode sssssosssenssessesessessseeserse 3 30 x Table of Contents Operating the detector in dual wavelength mode 3 31 Programming timed events threshold events and methods 3 34 PCN TAT SP E 3 42 Bororo yon As 1212 Beene eee E pa ide SE oe ence EE 3 42 Scanning new spectra 3 46 BIGUDE 3 SUBCLPIN e UD EE Ee 3 53 Getting information about stored spectrum eee etn phano ina kass 3 54 Reviewing a stored spectrum eser eiae nhi en XR eH RY SANE EVERY EN Nee 3 55 Subtract EE 3 55 Replay ite 8 SUSCI geet Eden 3 56 Scanning usine he UB DO c prote pe b LHP mele pope ER EE DH ptc bid 3 56 Scanning using a flow cell and a Syring E sssusa usenecie 3 59 Longer EE 3 60 Bnbur ng downs the E 3 62 4 Maintaining the Detector Queer fttt PxS eI rFDOM Gegend 4
10. Solvent miscibility Continued Boilin Miscibilit d Solvent Heal point c number i icu 1 atm M 5 2 Ethanol 1 20 78 3 14 210 5 3 Pyridine 0 94 115 3 16 305 5 3 Nitroethane 0 68 114 0 5 4 Acetone 0 32 56 3 15 17 330 5 5 Benzyl alcohol 5 80 205 5 13 5 7 Methoxyethanol 1 72 124 6 13 6 2 Acetonitrile 0 37 81 6 11 17 190 6 2 Acetic acid 1 26 117 9 14 6 4 Dimethylformamide 0 90 153 0 12 6 5 Dimethylsulfoxide 2 24 189 0 9 6 6 Methanol 0 60 64 7 12 210 7 3 Formamide 3 76 210 5 3 9 0 Water 1 00 100 0 How to use miscibility numbers Use miscibility numbers M numbers to predict the miscibility of a liquid with a standard solvent see Solvent miscibility on page C 3 To predict the miscibility of two liquids subtract the smaller M number value from the larger M number value Ifthe difference between the two M numbers is 15 or less the two liquids are miscible in all proportions at 15 C 59 F A difference of 16 indicates a critical solution temperature from 25 to 75 C 77 to 167 F with 50 C 122 F as the optimal temperature Ifthe difference is 17 or greater the liquids are immiscible or their critical solution temperature is above 75 C 167 F Solvent miscibility C 5 Some solvents prove immiscible with solvents at both ends of the lipophilicity scale These solvents receive a du
11. Window TaperSlit analytical cell UVlight The standard analytical inert and LC MS cells have a path length of 10 mm The semi prep and microbore cell path length is 3 mm The autopurification cell path length is 1 0 mm A variable path length flow cell path length 0 15 to 3 mm is also available Filtering noise The detector provides a Hamming filter to minimize noise The Hamming filter is a digital finite impulse response filter which creates peak height degradation and enhances the filtering of high frequency noise The behavior of the filter depends on the filter time constant you select You can program a filter time to be Fast Slow Normal or Other If you select Fast Slow or Normal you do not need to enter a value The filter constant is determined by the data rate If you select Other you can enter a value However the value you enter 1s rounded up or down to a value based on the data rate The filter time constant adjusts the filter response time to achieve an optimal signal to noise ratio Selecting Other and entering a value of 0 0 disables all filtering Principles of operation 1 7 1 8 Lower time constant settings produce these effects e Narrow peaks with minimal peak distortion and time delay Very small peaks become harder to discriminate from baseline noise Less baseline noise is removed Higher time constant settings produce these effects Greatly decrease baseline noise Shorten and broaden peak
12. rebuilding or replacing other flow cell components Required materials You need the following items to remove clean and replace the flow cell 1 4 inch flat blade screwdriver e Ethyl alcohol or methanol Flow cell rebuild kit Lint free swab Nitrogen e Plastic tweezers Powder free finger cots or gloves Torque wrench set to 0 904 N m 128 in oz or 8 in lb with 1 4 inch flat blade screwdriver bit Save all parts after removal unless otherwise indicated Most of the removed parts are needed when you reinstall a flow cell 4 8 Maintaining the Detector Removing the flow cell assembly To remove the flow cell assembly 1 Power off the detector and disconnect the power cord 2 After flushing and drying the flow cell see page 4 6 disconnect and cap the inlet and outlet LC tubing to the detector 3 Remove the front left hand panel cover 4 Using a 1 4 inch flat blade screwdriver loosen the three captive screws on the flow cell assembly front plate Flow cell assembly captive screws Maintaining the flow cell 4 9 5 Gently pull the assembly toward you 6 Once you remove the flow cell assembly from the detector chassis remove the cuvette subassembly from the flow cell assembly Detector flow cell assembly with the cuvette subassembly removed Flow cell assembly Cuvette subassembly 7 Place the flow cell assembly on a flat clean surface 4 10
13. 1 or 2 1 Filter 2 of 4 or Numeric Sec Slow Normal time constant 2 of 5 Normal Fast Other for Other A 0 0125 to 5 0 1 000 AA 0 5 to 5 0 0 to disable filtering Data out 3 of 4 Choice None Absorbance A Absorbance single i A Data out dual 3 of 5 Choice None Absorbance A Absorbance A Absorbance B A Maxplot A B Difference A B Ratio A B Auto zero on 3 of 4 or Check None Selected Checked inject 3 of 5 box Not selected Auto zero on 3 of 4 or Choice None _ To baseline To zero changes 3 of 5 To zero Disable Voltage offset 4 of 4 or Numeric mV Integer 2000 to O 4 of 5 2000 Chart polarity 4 of 4 or Choice None 4 of 5 _ Minimum AU 5 of 5 Numeric AU 0 0001 to 4 0000 0 1000 Minimum 5 of 5 Numeric None 0 00 to 999 99 0 00 ratio Maximum 5 of 5 Numeric None 0 00 to 999 99 2 00 ratio Using the operator interface 3 19 Operating the Trace and Scale functions Using the Trace function you can display an absorbance trace for the last n minutes as many as 60 of detector operation When you press the TRACE key the detector displays the absorbance acquired over the last 30 minutes by default It updates the trace once every 20 seconds When you press the Scale key Shift TRACE the detector displays the scaled trace with T1 ending time displayed 30 for the last 30 minutes by default You can change the ending time par
14. Anthracene 3 43 3 53 AUFS 3 44 cuvette 3 42 3 56 3 59 Erbium 3 44 flow cell 3 42 3 59 initiating 3 9 new spectra 3 46 3 53 pace 3 42 reference energy 3 45 replaying a spectrum 3 56 resolution 3 43 reviewing a scan 3 55 running a sample scan 3 50 sample energy 3 45 sample scan 3 45 3 49 screens 3 49 sensitivity 3 44 spectra 3 42 3 60 storing a scan 3 53 tick marks 3 44 timing 3 45 using the cuvette 3 56 3 59 zero scan 8 45 3 47 screens absorbance 3 3 3 4 HOME 3 3 secondary function screen 3 14 secondary functions 3 13 3 14 3 18 second order filter 1 3 1 4 1 10 selecting a location for the detector 2 3 Sensitivity icon 3 5 sensitivity Index 1 1 AUFS parameter 3 18 scanning 3 44 setting specification B 2 timed event parameter 3 35 serial number instrument 2 6 lamp 4 23 4 24 service diagnostic tests 5 11 5 19 Set absorbance diagnostic test 5 11 set voltage diagnostic test 5 11 setting detector up to run 3 13 fixed absorbance value 5 13 fixed voltage output 5 14 pulse periods 3 24 switches 5 15 shift icon 3 6 Shift key 3 10 shutting down the detector 3 62 signal connections 2 10 2 13 input 2 12 output 2 12 start of run 3 23 single pulse signal 3 24 single wavelength mode changing to dual 3 32 description 3 9 key 3 9 operating in 1 10 1 11 3 30 3 31 parameters 3 14 site requirements 2 3 slit entrance 1 4 solvent buffered solvents C 6 contamination 3 28 degasser 3 26 degassing 4 5
15. Corrective Action Select wavelengths that are each above or below 370 nm Error messages preventing operation During initialization calibration and operation the detector can display lt Error gt on the absorbance screen signifying a usually terminating malfunction and preventing further operation of the detector Error messages 5 5 Error display on the absorbance screen E 2 When you encounter such an error ensure that e no cuvette is in the cuvette holder and the empty holder is securely in place the flow cell is clean the front left hand panel door is shut securely Cycle power to the detector If the terminating error persists contact Waters Technical Service Instrument error messages Error Message Description Corrective Action Communication failure A D communication test 1 Cycle power to the Reference A D failed detector 2 Contact Waters Technical Service Communication failure A D communication test 1 Cycle power to the Sample A D failed detector 2 Contact Waters Technical Service Configuration not found Stored configuration Cycle power to the 2489 data are invalid Detector This action removes the error Dark current too high The dark energy level is 1 Cycle power to the nnnnnnn above 1000000 detector 2 Contact Waters Technical Service 5 6 Error Messages Diagnostic Tests and Troubleshooting Instrument
16. Displays the list of ROM momentary pulse to the options for generating and phart mark analog output A and or B manipulating spectra depending upon the current settings This key has no effect 1f chart mark is disabled on both channels Auto Zero Sets the AA From the absorbance iie absorbance offset so that the screen use this key to toggle AutoZero analog output A and or B between single and depending on the current dual wavelength modes The settings reads 0 AU You can current mode is indicated by enable or disable the an icon on the display auto zero function from the third absorbance screen see the figure on page 3 18 Run Stop Starts or stops Reset Resets the detector ne freezes the run clock The run clock to 0 minutes RUniStop elapsed time appears near the Returns the detector to initial conditions for the current method Arrow keys On screens with entry fields edit check box or choice list the border of the active field is thickened highlighted Using the arrow keys you move the highlighted board to the field you want to activate Up moves up or left Down moves down or right On screens with scrollable lists these keys move the highlighted border up toward the beginning of the list or down toward the end For other screens special instructions apply to the use of the Up and Down arrow keys for example the Display Contrast screen Using t
17. MaxPlot This mode results in the output of the larger of the two absorbance values scaled to the selected AUFS setting Use this mode when observing with one data channel multiple compounds that exhibit absorbancies at two separate wavelengths RatioPlot A B This mode produces the ratio of absorbance from two wavelengths Theoretically the ratio is constant for a pure chromatographic peak and variable for an impure peak which results in a nonsquared response Instead of a programmable AUFS the detector provides minimum and maximum ratio values that scale the ratio plot proportionally In addition a configurable minimum absorbance threshold activates ratio output scaling only when it reaches the absorbance at both wavelengths Difference Plot A B This mode plots the arithmetic difference in absorbance for the two monitored wavelengths Spectrum scanning When the detector is operating under the control of the Empower software the scanning function is disabled You can use the detector as a spectrophotometer to acquire spectra from either the flow cell or the cuvette You can scan and store as many as three spectra three reference or zero scans or three sample scans for playback or to compare with other spectra 1 12 Theory and Principles of Operation The major difference between the detector and a double beam spectrophotometer is that the detector uses only one flow cell or cuvette rather than a sample and
18. T Technical service 5 20 test peaks generating 5 17 The 4 15 theory of operation 1 1 1 14 threshold events clearing 3 41 programming 3 377 3 39 tick marks generating 3 44 time constant changing 3 13 function 3 16 timed event parameter 3 35 timed events and methods 3 34 3 42 clearing 3 41 deleting 3 37 description 3 35 parameters 3 35 3 36 programming 3 34 3 42 programming a new event 3 36 toggling between channels 3 4 tools to remove clean or replace flow cell 4 8 trace function 3 20 3 22 TRACE key 3 10 3 20 transient energy 5 21 troubleshooting contacting Waters 5 20 diagnostic tests 5 1 5 22 hardware 5 21 turning lamp off to conserve lamp life 3 60 3 62 turning lamp on or off from an external device 3 24 from front panel 3 11 manually 3 60 U unlock icon 3 6 unpacking the detector 2 5 Index 13 up down arrow keys 3 9 user selected diagnostic tests 5 9 5 19 using A B key 3 4 cuvette option 1 4 1 13 3 42 3 56 3 59 diagnostic tests 5 1 5 22 input and output diagnostic tests 5 12 keypad 3 7 3 12 RatioPlot function 3 33 sample and reference energy diagnostic tests 5 11 scale function to zoom 3 20 static flow cell for scanning 3 59 the 2489 as a spectrophotometer 1 12 V vacuum degassing See degassing validation kits 3 27 procedures 3 27 verifying detector 3 27 3 29 peak response 3 29 verification algorithm 1 9 viewing events within a method 3 40 voiding the warranty 4
19. Table of Contents Copyright Te TE ii Trademarks cannonu eee ees ope sess vus Eve ee pee Ve ate Us Da Ce E eR EV N EHE PEE KT S Ee TORRE TET ii Customer comments EE iii Contacting Waters oie ea rises sivi sies vidi es uiis iv Safety considerations ctr rtt X REED iv BAD naga uin eH HH bte epi ep Hp pipe p HM DEM rere v Operating this instrument ssssssesssessosssossossssoessossoossoossooseoosecoseooeocoeceseoseeceoeeoeese v udis siii MEM T v Audience and purpose v Intended use of the 2489 UV Visible detector ssseseesssessssssessssesssssssssssssssssses V RIDE M T T T ULM vi Ouais COREPOL oo ioo ba oo eei Eck ina Fea DRE EAA HE a A ce aH eration vi KR ET Te EE vi ISM Classification ISM Group 1 Class B aec iei rcp tr rere vi EC Authorized Representative c ecce esee esee nn nne n ane n haeo aen a sean eu vii 1 Theory and Principles of Operation RRE eere eee eene eee 1 1 Detector description 515 3 ettet stet aara e a e aeeoa ieinu aiiis 1 2 Principles OF operatiOt s ovre i ar ne ey Yee YS aa YER RAE 1 4 Detector ONS cine HE ee br EE 1 4 Wavelength vendication and test E 1 9 Oberational MAO EE 1 10 Single wavelength mode 1 10 iuslaravelensth vU us ea eo ees eeepc ae ass I dio Res 1 11 Pe POOLE BO MII cssc iuc Hx EE 1 12 E E 1 13 POE IOE deeg 1 14 EE 1 14 Table of Contents ix Thermal wander Mais femen aecenas iere ov dE Te Y TER EUER EE Ck E EA VP IK 14 VER 1 14 2
20. conserving lamp life 3 60 energy and performance 4 17 installing 4 21 4 23 lamp display and keypad diagnostic tests 5 11 5 15 monitoring aging 3 28 new 4 21 4 23 removing 4 18 4 21 replacing 3 28 4 16 4 23 serial number 4 23 4 24 timed event parameter 3 35 turning off 3 60 3 62 turning on or off 3 11 turning on or off from the separations module 2 16 turning on or off manually 3 60 use statistics 3 11 warranty 4 23 when to replace 4 18 Lamp key 3 11 lamp off icon 3 5 lamp on icon 3 5 line spikes 5 21 linearity specification B 2 locating the detector 2 3 lock icon 3 6 Lock key 3 11 locking the keypad 3 11 loss of current method conditions 3 41 M maintenance routine 4 5 maintenance considerations 4 2 manual calibration 3 11 3 30 manual lamp procedures 3 60 Index 8 mass spectrometer shock hazard A 4 maximum ratio field 3 33 function 3 16 parameter 3 19 MaxPlot function charting 3 15 features 1 14 obtaining 3 34 method active 3 40 choice list 3 10 current conditions 3 13 initial conditions 3 23 3 40 Method 3 34 3 40 preventing loss of current conditions 3 41 programming 3 34 3 42 resetting a stored 3 41 retrieving 3 40 storing 3 34 3 39 viewing events within 3 40 METHOD key 3 10 3 36 method number icon 3 34 minimum AU 3 15 3 33 minimum ratio field 3 33 function 3 15 parameter 3 19 miscibility of solvents C 3 C 6 mobile phase flushing 4 5 removing 3 62 mod
21. polarity timed event 3 35 primary 3 18 ratio minimum AU 3 19 sample and zero scan 3 47 sample scan 3 45 3 49 secondary 3 18 sensitivity timed event 3 35 single wavelength mode 1 10 1 11 SW1 timed event 3 35 SW2 timed event 3 36 time constant timed event 3 35 Index 9 timed event 3 35 voltage offset 3 19 wavelength 3 18 wavelength timed event 3 35 zero scan 3 45 peak response test 3 29 peaks generating test 5 11 5 17 performing verification procedures 3 27 3 29 photodiodes 1 4 plumbing connections 2 6 2 8 Plus minus key 3 12 polarity timed event parameter 3 35 polarity chart 3 13 power requirements 2 4 surges 5 21 powering off the detector 3 62 preventing loss of current method conditions 3 41 Previous key 3 10 primary functions 3 14 3 18 principles of operation 1 1 1 14 programming absorbance threshold events 3 37 3 39 switches 3 24 threshold events 3 37 3 39 timed events and methods 3 34 3 42 pulse periods setting 3 24 purpose and audience v Q qualification using the cuvette 1 3 R ratio minimum AU parameter 3 19 RatioPlot function charting 3 15 Index 10 comparing analytes using 1 14 maximum ratio 3 16 minimum AU 3 15 minimum ratio 3 15 obtaining 3 33 rear panel fuses 4 25 illustration 2 9 signal connections 2 10 reassembling the flow cell 4 8 4 16 rebuild kit flow cell 4 11 rebuilding the flow cell 4 14 4 15 recalling the absorbance screen using the HOME key 3 4
22. rectangular wave To define the pulse period or the frequency of a wave see Configuring the detector on page 3 22 To program a threshold event 1 2 Press the METHOD Shift A B key on the detector keypad Press 2 Threshold events Threshold events screen VA Absorbance A Above 0100 Ja Set set switch 1 l To on Press Enter to advance to the next Set field or press the s and t keys to move among the three fields on the threshold events screen 3 38 Preparing the Detector 6 When the Set field is active press Enter to display the threshold events choice list or press the number corresponding to the event you are programming see the table above When the To field is active press Enter to display the options in the table shown above or press the number corresponding to the threshold parameter you are programming To set the threshold on the other channel press A B Storing a method A method consists of all programmable parameters on the absorbance and associated screens as well as timed and threshold events You can store the current method by selecting a location from 1 to 5 To store a method 1 Press METHOD Shift A B to return to the Method choice list see the figure on page 3 36 Press 4 Store method Result A method number field appears Storing a method method number field mem method store mi number 5 Reset mem Caution No warning
23. see Waters Licenses Warranties and Support Services Site selection and power requirements 2 5 Tip Make sure the instrument serial number found on the nameplate on the rear panel or inside the front left panel of the detector corresponds to the number on the instrument integrity certificate Making plumbing connections Before initially starting the detector Complete the plumbing connections described in this section Complete the electrical connections see Making electrical power connections on page 2 9 Caution Observe Good Laboratory Practices when handling solvents Refer to the Material Safety Data Sheets for the solvents in use Requirement You must make the following plumbing connections on your detector unit Column connections Drip management system connections Recommendations Before completing the column connections perform the verification procedures described in Verifying that the detector operates properly on page 3 27 Install a waste reservoir connected to the drain tube and located next to the rubber foot on the front lower left hand section of the detector Use Tygon tubing to connect the drain tube to the waste reservoir Caution The detector as shipped has a standard analytical flow cell pressure rated at 6895 kPa 69 bar 1000 psi To prevent damage do not connect any tubing or device that can cause backpressure to exceed the pressure rating of the tubing
24. 0 9 0 through 9 into a current specific shifted numeric keys field Also positions the cursor at the corresponding entry in a list 0 tenth item Selects the corresponding number from a choice list 3 10 Preparing the Detector Detector keypad description Continued Key Unshifted Shifted 1 See 0 9 above Lamp Displays a lamp use ECH statistics for the currently 1 installed lamp and provides the means for you to turn the lamp on or off The current state of the lamp is indicated by an icon on the absorbance screen 2 See 0 9 above Lock Use from the Lock absorbance screen to lock or 2 unlock the keypad The lock prevents inadvertent changes to detector settings The current lock state is indicated by an icon on the absorbance Screen 3 See 0 9 above Calibrate Initiates Calibrate wavelength calibration 3 4 See 0 9 above System Info Displays system information including firmware version and instrument serial number System Info 4 6 See 0 9 above Contrast Permits contrast adjustment viewing angle of 6 the liquid crystal display Contrast 0 See 0 9 above Cancel In some modes backs out of a prompt without 0 completing the task The word Cancel appears as a cue in the lower right hand border of the message text Cancel Using the operator inter
25. 16 replacing parts 4 5 4 16 scanning 3 42 3 59 specifications 4 15 B 5 static 3 59 tools required to remove clean or replace 4 8 window 4 13 flow cell assembly removing 4 9 flow cell rebuild kit 4 11 flushing buffered mobile phase 4 5 flow cell 4 6 fraction collector connecting to 2 33 functions absorbance 3 14 analog outputs dual wavelength 3 15 analog outputs single wavelength 3 14 auto zero 3 17 auto zero 3 17 auto zero on wavelength changes 3 17 dual wavelength mode 3 31 filter time constant 3 16 MaxPlot 3 15 3 34 primary 3 14 RatioPlot 3 15 3 33 scale 3 20 3 22 secondary 3 14 time constant 3 16 trace 3 20 3 22 zoom 3 20 fuse holder 4 25 Index 6 replacing 4 25 4 26 G gas solubility C 7 C 8 generate test peak diagnostic test 5 11 generating auto zero on inject from the 2695 2 17 chart mark from the 2695 2 18 chart marks 2 27 3 9 spectra 3 9 test peaks 5 17 tick marks 3 44 grating diffraction 1 4 H handling symbols A 13 help key 3 9 3 25 HOME key 3 4 3 9 3 13 HOME screen See absorbance screen l T O signals 2 12 icons absorbance 3 5 channel on 3 5 channel selector 3 5 keypad lock 3 6 keypad unlock 3 6 lamp off 3 5 lamp on 3 5 method number 3 34 next 3 6 run time 3 6 Sensitivity 3 5 shift 3 6 sticky diagnostic tests 3 6 table of 3 5 wavelength 3 5 wrench 3 6 idle mode 3 3 inaccurate peak areas 3 32 initial method conditions 3 9 3 23 3 40 initializing th
26. 20 Acquiring data and controlling the detector with Empower software by using the e SAT IN module instead of the Ethernet bus requires connections between the following hardware Laboratory acquisition and control environment LAC E module LAC E Acquisition Server or busLAC E card Ethernet satellite interface e SAT IN module e SAT IN module The Waters e SAT IN module shown in the figure below translates analog signals from devices such as the detector into digital form It then transmits these digital signals to the busLAC E or LAC E card installed in the Empower workstation Installing the Detector e SAT IN module front panel Power switch Waters e SAT IN e Me CHANNEL1 CHANNEL 2 Analog inputs I O connector LEDs To connect the detector to the Empower workstation Caution The e SAT IN module does not have a power switch Always disconnect the power cord at either the wall outlet or the power supply before attaching or removing the power connection to the e SAT IN module To ensure proper startup of the e SAT IN module do not power on the module until you perform all procedures in the Waters e SAT IN Module Installation Guide Improper startup can damage the unit and void the warranty Connect the detector to the e SAT IN module See page 2 21 Connecting the detector to the e SAT IN module The e SAT IN module connects to the detec
27. 23 voltage offset function 3 13 3 16 parameter 3 19 VW warming up the detector 3 28 warning symbols A 2 A 5 warranty lamp 4 23 voiding 4 23 Waters 600 Series Pump Index 14 auto zero connections 2 29 chart mark connections 2 30 configuring the detector lamp signal 2 28 connecting to 2 28 2 31 Inject Start connections 2 31 Waters 717plus Autosampler auto zero connections 2 32 connecting to 2 31 2 33 Inject Start connections 2 32 Waters 745 745B 746 Data Module connecting to 2 23 2 25 Waters data system control 2 13 Waters TaperSlit flow cell description 1 6 1 8 exploded view 4 13 frontal view 4 11 specifications 4 15 B 5 Waters Technical Service contacting 2 5 4 2 5 20 wavelength accuracy specifications B 2 calibration 3 11 3 30 changes auto zero on 3 17 changing 3 9 3 32 ending 3 42 functions in dual 3 31 icon 3 5 parameter 3 18 range specifications B 2 selection C 9 C 11 starting 3 42 timed event parameter 3 35 wrench icon 3 6 Z zero scan definition 1 13 parameters 3 45 3 47 running 3 47 screens 3 49 zoom function 3 20 3 51 Index 15 Index 16
28. 230 1000 2000 300 400 10 Azo NZzN 285 400 3 25 C 12 Solvent Considerations Electronic absorption bands of representative chromophores Continued Chemical max max max max Chromophore e configuration nm L m cm nm L m cm Nitroso N O 302 100 Nitrate ONO92 270 12 shoulde r Allene C C g 210 230 21 000 acyclic Allene C C 3 260 35 000 Allene C C 4 300 52 000 Allene C C 5 330 118 000 Allene C C 9 230 260 3000 8000 alicyclic Ethylenic C C C C 219 6 500 Acetylenic Ethylenic C C C N 220 23 000 Amido Ethylenic C C C 0 210 250 10 000 Carbonyl 20 000 Ethylenic C C NO9 229 9 500 Nitro Wavelength selection Willard H H and others Instrumental Methods of Analysis 6th ed Litton Educational Publishing Inc 1981 Reprinted by permission of Wadsworth Publishing Co Belmont California 94002 C 13 C 14 Solvent Considerations Index Symbols key 3 12 key 3 9 3 25 e key 3 12 Numerics 2695 separations module connecting to 2 17 2 19 generating a chart mark from 2 18 generating an auto zero on inject from 2 17 starting a method from 2 15 turning the lamp on or off from 2 16 600 Series Pump See Waters 600 Series Pump A A B key 3 4 3 10 absorbance difference plot 3 16 function 3 14 halted by fatal error 5 5 icon 3 5 MaxPlot function 3 15
29. 3 34 and the table on page 3 35 for more information on programming the lamp to turn on or off using a timed event See Configuring event inputs contact closures on page 3 23 for more information on programming the lamp through the external contact closure Shutting down the detector If you need to power off the detector for an extended length of time you must remove any buffered mobile phase from the fluid path Caution To avoid damage to the column remove it before you perform the following procedure Before you remove the column see your column care and use guide Removing buffered mobile phase To remove mobile phase from the fluid path of the detector 1 Replace the buffered mobile phase with 100 HPLC quality water and flush the system for 10 minutes at 3 mL min 2 Replace the 10096 water mobile phase with a solution of 90 10 methanol water and flush the system for 10 minutes at 3 mL min Follow the recommended procedures for injector purging and priming for the pump in use with your HPLC Shutting down the detector To shut down the detector press the On Off switch 3 62 Preparing the Detector Maintaining the Detector Contents Topic Page Contacting Waters technical service 4 2 Maintenance considerations 4 2 Proper operating procedures 4 4 Maintaining the flow cell 4 5 Replacing the lamp 4 16 Replacing fuses 4 25 4 1 Contacting Waters technical service If you are located in the US
30. 5 6 Chart mark Hold Inject 1 6 7 Chart mark Hold Inject 1 7 8 Ground Hold Inject 2 8 9 Auto Zero Hold Inject 2 9 10 Auto Zero Ground Chart Out 11 Chart Out Connecting to other devices You can connect the detector to a many HPLC system devices This section describes how to connect the detector to the following devices eSAT IN module instead of Ethernet e Waters 745 745B 746 Data Module Chart recorder e Waters 600 series pump Waters 717plus autosampler Waters fraction collector For details on connecting to other data modules see the operator s guide for the module you are using Connecting to other devices 2 19 Required materials When connecting cables to the terminals on the rear panel of the detector you need the following tools Small flat blade screwdriver Electrical insulation stripping tool Connecting signal cables To connect cables from other HPLC system devices to the and II terminals on the rear panel of the detector 1 Remove terminal I or II see page 2 12 2 Unscrew the connecting pin terminal 3 Using the stripping tool strip the wire about 3 mm 1 8 inch from the end Insert the stripped wire into the appropriate connector Tighten the screw until the wire 1s held securely in place Reinsert the terminal Sch og Ol oo Press firmly to ensure that it is inserted fully Connecting the detector to Empower using an e SAT IN module 2
31. An active field containing a choice list is indicated by a numeral to the right of the field and within the thickened border 3 12 Preparing the Detector 4 To display a choice list press Enter Then take one of these actions e Press the corresponding number key to select an item immediately Use the up and down arrow keys to scroll through the list and then press Enter Tip You can press the numeral that corresponds to a desired choice without first pressing Enter Rule The Up and Down arrow keys do not incrementally increase or decrease numerical field entries Use the numerical keypad for that purpose Navigating to and from the absorbance screen Pressing HOME from most screens causes the absorbance screen to appear From the absorbance screen you can access several secondary functions To move to the secondary function screens of the absorbance screen press Next These are the secondary functions Analog output specifications time constant Absorbance offset Voltage offset Chart polarity Enable disable inputs e Enable disable external events Tip The parameters you enter into the secondary function fields become part of the current method conditions and are stored when you store the method see page 3 34 In single wavelength mode the detector displays three additional screens labeled 2 of 4 3 of 4 and so on In dual wavelength mode the detector displays four additional screens labele
32. Analog 1 Analog 1 Ground Analog 2 Analog 2 Switch 1 Switch 1 Ground Switch 2 0 Switch 2 745 745B 746 terminals SP OO OO JO Om P GO A 2489 connector Analog 1 Analog 1 Ground Analog 2 Analog 2 Switch 1 Switch 1 Ground Switch 2 0 Switch 2 745 745B 746 terminals A OO OD JO Om oh A Connecting the detector to a chart recorder Recorder signal The A and B terminals on the rear panel of the detector provide 2 V analog output signals that you can use to connect the detector to a chart recorder Connecting to other devices 2 25 To send 2 V signals from the detector to a chart recorder use the cable provided in the 2489 Detector Startup Kit to make the connections summarized in the table and illustrated in the figure that follows it Detector connections to a chart recorder 2489 detector B Inputs Chart recorder and outputs terminal Pin 1 Analog 1 Pin 3 Ground Pin 4 Analog 2 Pin 8 Ground To minimize the chance of creating a ground loop that can adversely affect measurement connect the shield of the cable to the chassis ground at one end only For connection to other data systems a cable can help remove the incidence of ground loops Tip The detector 1s optimized for use with the 2 V analog output Connect the detector to a chart recorder using the 2 V analog
33. Marker II Pin 7 Chart mark Pin 9 Event Marker I Pin 6 SW1 Pin 7 External Count In I Pin 8 Ground Pin 8 External Count In 2695 separations module 717plus autoinjector Inject Start External Start In Inject Start External Start In See also Documentation provided with your fraction collector for complete 2 34 Installing the Detector Preparing the Detector Contents Topic Page Initializing the detector 3 2 Using the operator interface 3 4 Scanning spectra 3 42 After you install the detector you can set it up and operate it as a stand alone instrument or as part of a an Empower or MassLynx data system Asa stand alone instrument You can use the detector as a stand alone detector within a system or with any fluid handling unit injector integrator or data system You can program the front panel of the detector for stand alone operation except when it is in remote mode see page 3 26 As part of an Empower or MassLynx system Configure the detector with either data system to control and collect digital data To do so follow the instructions in the system s online Help Requirement To ensure accurate operation and before pumping mobile phase or solvent through the flow cell be sure to perform the procedures on page 3 27 Initializing the detector 3 2 Before you power on the detector be sure the connector from the rear panel to the power source 1s properly in
34. a reference pair Recommendation Use a matched pair of cuvettes for the zero and sample scans The detector obtains an absorbance spectrum by performing two types of scans on the flow cell or using the cuvette Zero scan Characterizes the baseline absorbance spectrum of a solvent Sample scan Subtracts the zero scan so the results displayed or charted are of the sample only To obtain a spectrum of a sample using the detector run a zero scan first followed by a sample scan Typically you run the zero scan using pure solvent The sample scan is a scan of the analyte dissolved in that solvent Spectra can be simultaneously charted on the channel A output or acquired and stored in memory for later playback See also Scanning using the cuvette on page 3 56 and Scanning using a flow cell and a syringe on page 3 59 Cuvette operations The detector cuvette option is used to measure the absorbance spectrum of a sample in a cuvette To generate and store a spectrum 1 Acquire a zero scan which measures the absorbance of the contents of the cuvette and flow cell over the desired wavelength range 2 Acquire a sample absorbance scan which measures the absorbance of the analyte dissolved in mobile phase The detector subtracts the zero scan from the sample scan to create a sample spectrum Since the cuvette scan is acquired by measuring the absorbance from a light path that includes both the flow cell a
35. an integral part of a Waters chromatography system The detector can be configured with Empower or MassLynx software systems 1 2 Theory and Principles of Operation The detector has the following capabilities Stand alone programmability Stores as many as five user defined programs or methods consisting of as many as 50 programmable timed events and two threshold events each Single or dual wavelength Monitors absorbance at one or two discrete wavelengths Wavelength verification reference filter Ensures wavelength accuracy Automatic second order filter Automatically engaged for wavelengths of 370 nm and greater and removed for wavelengths 369 nm or less Spectrum scan and storage Supports spectrum scan display subtraction storage and playback in addition to standard absorbance and UV Vis functionality Cuvette qualification Facilitates qualification of the detector by insertion of a standard in a cuvette without breaking any plumbing connections Waters qualification kits available in cuvette form support this feature which allows the detector to serve as a benchtop spectrophotometer Cuvette sample analysis Allows recording of the spectrum of any sample placed in the cuvette Method editing and storage Supports basic method programming storage and retrieval from the front panel Full diagnostic capability Supports built in diagnostic tools to optimize functionality an
36. di tubi di polimero pressurizzati Spegnere tutte le fiamme vive nell ambiente circostante Non utilizzare tubi eccessivamente logorati o piegati Non utilizzare tubi non metallici con tetraidrofurano THF o acido solforico 0 nitrico concentrati Tenere presente che il cloruro di metilene e il dimetilsolfossido provocano rigonfiamenti nei tubi non metallici riducendo notevolmente la pressione di rottura dei tubi stessi Advertencia se recomienda precauci n cuando se trabaje con tubos de pol mero sometidos a presi n El usuario deber protegerse siempre los ojos cuando trabaje cerca de tubos de pol mero sometidos a presi n Si hubiera alguna llama las proximidades No se debe trabajar con tubos que se hayan doblado o sometido a altas presiones Es necesario utilizar tubos de metal cuando se trabaje con tetrahidrofurano THF o cidos n trico o sulf rico concentrados Hay que tener en cuenta que el cloruro de metileno y el sulf xido de dimetilo dilatan los tubos no met licos lo que reduce la presi n de ruptura de los tubos TEE B Bm THAO MERI ADEE P AER EE BOURSE JJ RS AAT RUF re SE TR S o TE BASEL IU B C o AN EAE ROB eg BCE AN BFE AE SES Be PA UU BET AAR sat Foe HE aR mg ENC id PS V ierg ur Bauer 3 SN IE SS EP AU Ne KARE REI T RS REJ o i 1 E Safety Advisories Es ARTI EH
37. either test press DIAG and then press 5 Other diagnostics Other diagnostics choice list Generate test peaks z Optical Filter override Previous choices lt lt To generate test peaks 1 From the Other diagnostics choice list press 1 Generate test peaks to generate test peaks every 100 seconds on the chart trace or other output Restriction The Generate test peaks diagnostic test works only in single wavelength mode Every 100 seconds the detector generates an approximate 1 AU peak with a standard deviation of 10 seconds on the trace chart or data system display until you disable the Generate test peaks diagnostic test The amplitude of the test peaks is affected by your choice of filter time constant This is a sticky diagnostic test When this routine is selected the choice list changes to read Disable test peaks 2 Press 1 Disable test peaks from the Other diagnostics choice list to turn off this diagnostic test 5 18 Error Messages Diagnostic Tests and Troubleshooting To override the optical filter 1 From the Other diagnostics choice list press 2 Optical filter override to manually override the detector s automatic filter choice Optical Filter Override diagnostic test display Optical Filter mt Override mode Automatic is the normal made af operation In the Optical Filter Override diagnostic test display press Enter to display the fo
38. flow cell Recommendation To prevent reabsorption of dissolved oxygen for systems using vacuum degassers run the solvent degasser continuously when operating the detector at wavelengths less than 230 nm 2 8 Installing the Detector Making electrical power connections To connect the detector to the AC power supply 1 Plug the receptacle end of the power cord into the AC input connector on the rear panel of the detector see the figure below 2 Plug the other end of the power cord into a properly grounded AC power source Detector rear panel The detector connects to other Waters components through rear panel electrical connections Detector rear panel electrical connections O moo Bold Inputs and moog moog D outputs S S E I 8 62626262 O605006596969060 Nu X 980506000 A RS 232 re 2 LJ EI Hz 50 60 I Made in USA O E Sa 9 rh ETHERNET Power input Fuse holder Rear panel connections enable the following signals Analog outputs There are two pairs of attenuated analog channel outputs Each pair supports 2 V output to external devices or data systems They are labeled I and II For input output voltage current specifications see page B 2 The 2 V output for I and II is scaled based on the AUFS absorbance units full scale setting for each channel The detector does not Making elec
39. for a normal chromatogram or for an inverted chromatogram This function changes the direction of the plot on the 2 V output similar to reversing the leads to an external chart recorder 1 10 Theory and Principles of Operation Filter time constant Programs a filter time in seconds Options are Fast Slow Normal or Other If you select Fast Slow or Normal you do not need to enter a value The filter constant is determined by the data rate If you select Other you can enter a value but the value you enter is rounded up or down to a value based on the data rate Selecting Other and entering a value of 0 0 disables all filtering Analog rate Specifies a value as many as 80 Hz Secondary parameters Pressing Next on the absorbance or HOME screen while in single wavelength mode causes several pages of these secondary or less frequently specified parameters to appear Absorbance offset in mV Auto zero on inject Auto zero on changes Primary and secondary functions on page 3 14 and the table on page 3 5 explain the functions ranges and defaults of these parameters Dual wavelength mode In dual wavelength mode the detector can monitor two wavelengths one on channel A and one on channel B The sampling frequency is reduced to 1 or 2 Hz limiting use of this mode to more standard chromatography where peaks span at least 20 seconds to enable full characterization of a peak You can use dual wavelength mod
40. interference second order effects you can observe additional peaks and inaccurate peak areas 3 32 Preparing the Detector Dual wavelength 370 nm threshold warning message Wouwe lengths span 3r nm Order Filter mot in uze For 370 nm Obtaining a RatioPlot The RatioPlot output on one channel only channel A depends on the values you specify on absorbance screen 5 for minimum and maximum ratios You must be operating the detector in dual wavelength mode to obtain a ratio plot The RatioPlot provides a plot of the ratio of the absorbances of two wavelengths from 0 V to 2 V The minimum and maximum ratio parameters are measured ratio units not absorbances See page 3 14 To obtain a RatioPlot 1 Besure the detector is operating in dual wavelength mode see the previous discussion From the absorbance screen press Next to reach screen 3 of 5 In the Data out field press 8 ratio A B Press Enter to select the ratio plot Press Next until screen 5 of 5 appears OF e qe eoo ibe Enter the minimum AU and then press Enter Tip The minimum AU field contains a threshold value The RatioPlot function does not plot if either wavelength does not exceed the minimum AU threshold 7 Enter the minimum ratio for the RatioPlot and then press Enter 8 Enterthe maximum ratio for the RatioPlot and then press Enter 9 Return to the absorbance screen by pressing the HOME key Using the operator interface 3 33
41. message appears when the method number you select is already assigned to a previously stored method Pressing Enter stores the current method conditions overwriting any previous method stored in the same slot Using the operator interface 3 39 3 Enter a number from 1 to 5 and press Enter Result A brief message Storing as method n appears 4 When the display returns to the Method choice list the method number you selected appears within the method icon That method remains active until you retrieve another method or reset the detector to default conditions Method Retrieving a method To retrieve a previously stored method 1 Return to the Method choice list by pressing METHOD Shift A B 2 Press 3 Retrieve a method Result The last method number stored or retrieved appears in the method number slot box 9 Enter the number of the method you want to retrieve and press Enter Result A brief message Retrieving method n appears When the Method choice list reappears the method number icon incorporates the method number you specified see the table on page 3 5 Viewing events within a method To view timed and threshold events that make up a stored method 1 Retrieve the method see page 3 40 Result Once you specify a method number to retrieve the method choice list appears and the method number appears within the method number icon 2 Press 1 to view the timed events or 2 to view thre
42. mode To change from single to dual wavelength mode 1 From the absorbance or HOME screen when in single wavelength mode the wavelength icon shows press the 4 AA key Shift Auto Zero Result A momentary message Setting up dual wavelength mode appears Enter the wavelength to be monitored in the A field and then press Enter Enter the other operating parameters and timed or threshold events if desired Press the A B key to switch channels The absorbance screen for the other channel appears Enter the operating parameters for monitoring the second wavelength including timed and threshold events if desired If both selected wavelengths are greater than 370 nm 1 nm the detector applies the second order filter to block unwanted UV light If both selected wavelengths are less than 370 nm 1 nm the detector removes the second order filter If the selected wavelengths bracket the 370 nm 1 nm threshold the detector does not apply the second order filter and issues a warning message that any data collected for the wavelength above 370 nm can contain inaccuracies because of possible UV light interference second order effects Recommendation In dual wavelength mode select a wavelength pair below or above 370 nm If one or both of the selected wavelengths spans the 370 nm threshold the detector beeps three times and the warning message shown below appears Because of possible UV light
43. module connections to the detector for auto zero on inject 2695 connector B WW N Inject Start Inject Start Ground Stop Flow Stop Flow Hold Inject 1 Hold Inject 1 Hold Inject 2 Hold Inject 2 Ground Chart Out Chart Out A OD OD d OO P Gab A CSS MONN 2489 connector II Inject Start Inject Start Ground Lamp On Lamp On Chart mark Chart mark Ground Auto Zero 0 Auto Zero A DO DD JO Om P Go h Generating a chart mark on injection To generate the chart mark function on the detector at the start of an injection make the connections summarized in the table and illustrated in the figure that follows it 2695 separations module B inputs and outputs Pin 1 Inject Start 2489 detector Il Pin 6 Chart mark Pin 2 Inject Start Pin 7 Chart mark Before you can generate a chart mark from the separations module you need to configure the chart mark signal at the detector s front panel The default chart mark signal setting is Low see the discussion page 3 23 for more information Installing the Detector Separations module connections to the detector for making a chart mark on injection 2695 connector B 2489 connector II 1 Inject Start Inject Start 1 2 Inject Start Inject Start 2 3 Ground Ground 3 4 LampOn Stop Flow 4 5 Lamp On Stop Flow
44. noncondensing Shipping and storage temperature Shipping and storage humidity 30 to 60 C 0 to lt 95 noncondensing Electrical Specifications Line frequency 50 to 60 Hz Line voltage 100 to 240 Vac Max VA input 185 VA Operational specifications B 3 Operational specifications Continued Condition Fuse ratings Specification Two fuses 100 to 240 Vac 50 to 60 Hz F 3 15 A 250 V fast blo 5 x 20 mm IEC Attenuated analog output channel 2 VFS Attenuation range 0 0001 to 4 000 AU 2 V output range 0 1 to 42 1 V Two event outputs Type Contact closure Voltage 30 V Current 1 A Four event inputs Input voltage 30 V maximum 100 ms minimum period Dimensions Height 20 8 cm 8 2 inches Length 50 3 cm 19 8 inches Width 28 4 cm 11 2 inches Weight 9 3 kg 20 5 pounds Optical specifications B 4 Optical specifications Condition Monochromator type Specification Fastie Ebert Grating Plane holographic 1800 grooves mm Optical bandwidth 5nm Lamp power 30 W Specifications Optional Waters TaperSlit flow cell specifications Optional Waters TaperSlit flow cell specifications Sample tube Volume Path internal Pressure length ul mm diameter rating Inches In Out psi bar Analytical cell 10 10 0 009 0 009 1000 70 Se
45. of full apply scale 5 Polarity E Yes 2 6 Auto zero Does not Does not apply Yes apply 7 Lamp 1 Off Off No 2 On 8 Switch 1 1 High High No 2 Low 3 Pulse Using the operator interface 3 35 Timed event parameters Continued Number Event Units Range or default SES channel 9 Switch 2 1 High High No 2 Low 3 Pulse 4 Rect wave 10 Threshold AU 4 0000 to 4 0000 or Yes variable depending on output selection To program a new timed event 1 Press the METHOD Shift A B key on the detector keypad Method choice list Timed events z Threshold events 3 Retrieve a method 4 Stare method 2 Press 1 Timed events 3 Enter the time for the event Timed events screen Week Event 1 of 1 ar 25 00 Jin set wavelength H Te 299 Je 4 Press Enter to enter the time Tip To advance to the Set field Events choice list press the t key 3 36 Preparing the Detector 5 Press Enter again to display the choice list or if you know the event number press the number for the event you are programming 6 Enter the appropriate wavelength in nanometers in the To field if that field appears Requirement To program the same event on both channels you must enter two events one for channel A and one for channel B 7 Press A B to set the threshold on the other channel Tips ON Aor ON B indicates the channel the event is programm
46. or flow cell 2 6 Installing the Detector Connecting columns The line connections to the detector are located in the front right hand side of the flow cell assembly see the figure below To make the inlet and outlet tubing connections 1 Attach the stainless steel compression fitting and ferrule supplied in the startup kit 2 Connect the inlet tubing to the column outlet Ensure the tubing is seated firmly and then tighten the compression screw 3 Connect the Tygon tubing to the flow cell outlet tubing and route the tubing to a waste container Plumbing connections Making plumbing connections 2 7 Assembling the fittings To assemble each fitting 1 Slide the compression screw over the tubing end followed by the ferrule 2 Mount the ferrule with its tapered end facing the end of the tubing Ferrule and compression screw assembly Compression screw Tubing end cut straight and smooth to achieve maximum column efficiency Tube Ferrule Distance determined by each application such as union or column fitting Making connections To make connections at the column outlet and detector inlet and at the detector outlet 1 Seat each tubing end in the appropriate fitting 2 Seat each ferrule by tightening the compression screw 1 2 turn past finger tight Requirement To ensure accurate verification power on the detector before pumping any mobile phase or solvent through the
47. pace from the most recent zero scan Only when those values are identical for both the zero and sample scans can the zero scan be subtracted You use the SCAN key Shift Chart mark to run a new zero or sample scan store review subtract and review and replay stored or existing scans During a sample scan data are charted via analog channel A using the specified AUFS setting At the same time sample energy at 150 nA V is charted via channel B During a zero scan data are charted via analog channel A At the same time reference energy at 150 nA V is charted via channel A at the AU specified on channel A Scanning spectra 3 45 Scanning new spectra To specify a new spectrum 1 Press SCAN Shift Chart mark A Scan choice list appears Scan choice list New con z Store last scan 3 Det scan info d Review Press 1 New scan or use the s and t keys to move through the Scan choice list Result The detector displays the first of three parameter screens for a sample scan or four parameter screens for a zero scan figure Zero and sample scan screen on page 3 49 Press Next to advance through the New scan parameter screens On the first New scan screen specify the type of scan Press 1 for sample scan or press Enter to display the choice list The detector displays two additional screens Press 2 for zero scan or press Enter to display the choice list The detector displays three addition
48. power on the unit Tip The instrument firmware automatically delays operation for 5 minutes to allow the lamp to warm after it has been reignited 4 22 Maintaining the Detector Aligning the lamp Cutout in 1 00 position Recording the new lamp serial number Caution Always run the Change Lamp diagnostic test after you install a new lamp see Using the lamp display and keypad diagnostic tests on page 5 15 f you do not record the new lamp serial number following the procedure in this section the lamp warranty is voided The detector software records and stores the serial number and date of installation of a new lamp so that you can periodically check its age and its number of ignitions To record the new lamp serial number 1 When the detector warms up press the DIAG key 2 Press 4 Lamp display amp keypad 3 Press 1 Change lamp Tip Be sure to enter the 9 digit lamp serial number and not the lamp part number when performing this procedure Replacing the lamp 4 23 4 Enter the 9 digit serial number of the new lamp in the active field Change lamp screen Serial number of lamp 123456789 Install date ear Hours im Bios 0 5 5 Press Enter to store the serial number and to move to the Install date field 6 Select the month from the choice list and press Enter twice to update the month and to select the next field which specified the day 7
49. secured in place Caution Do not touch the glass bulb of the new lamp Dirt or fingerprints on the bulb adversely affect detector operation If the lamp needs cleaning gently clean the bulb with ethanol and lens tissue Do not use abrasive tissue or apply excessive pressure Replacing thelamp 4 21 Before you begin 1 Unpack the lamp from its packing material Note The new lamp s features can vary slightly from those shown in the figure 2 Inspect the new lamp for particles or dirt If necessary clean the lamp with a gas duster or lens tissue 3 Record the serial number located on a label attached to the lamp connector wire using the procedure in Recording the new lamp serial number on page 4 23 Requirement Make sure the detector is powered off and the power cord is disconnected To install the new lamp Caution When you change the lamp always power off the detector After installing a new lamp power on the detector and always wait at least 5 minutes for the new lamp to warm up 1 Position the lamp so the cut out located on the lamp base is at the 1 o clock position see the figure below in line with the alignment pin on the lamp housing There is no additional alignment required Gently push the lamp forward until it bottoms into position Tighten the two captive screws Reconnect the lamp s power connector Qv dem eo ei When ready to resume operation of the detector reconnect the power cord and
50. the detector see figure below Two fuses are shipped in place with the detector rated as indicated in Power requirements on page 2 4 Detector rear panel fuse holder O n n nu un BBBBBBBBEB n BBBBBBHBBB cz OH LJ Mn 0 O VA_ 145 gt F 3 15A 250V Q ETHERNET Fuse holder Warning To avoid electric shock power off and unplug the instrument before inspecting the fuses Replacing fuses 4 25 The detector requires two 100 to 240 VAC 50 to 60 Hz F 3 15 A 250 V fast blo 5 x 20 mm IEC fuses Suspect a fuse is open or otherwise defective when the detector fails to power on the fans do not operate To replace the fuses Requirement Replace both fuses even when only one is open or otherwise defective 1 Power off the detector and disconnect the power cord from the power entry module 2 Pinch the sides of the spring loaded fuse holder which is above the power entry module on the rear panel of the detector 3 With minimum pressure withdraw the spring loaded fuse holder Removing and replacing the rear panel fuses and fuse holder Fuse holder 4 Remove and discard both fuses 5 Make sure that the new fuses are properly rated and then insert them into the holder and the holder into the power entry module gently pushing until the assembly locks into positio
51. the mobile phase or solvent in which your sample is dissolved 2 Runa zero scan according to the procedure on page 3 46 3 With a syringe fill the flow cell with the analyte and then run a sample scan according to the procedure on page 3 46 Use the storage review subtract and review and replay functions of the detector to compare the scanned data Conserving lamp life You can conserve the lamp without powering off the detector by leaving the instrument on and turning off only the deuterium lamp Tip If the detector is operating under remote control you can program the controller to turn the lamp off and on without using the front panel Recommendation Waters recommends that you program the lamp to shut off or turn the lamp off manually only if the value of the Lamp off parameter is more than 4 hours Without powering off the system you can conserve lamp life by turning power to the lamp off and on again manually programming a timed event to turn the lamp off and on again programming the lamp to turn off and on again using the external contact closure To turn power to the lamp on and off manually use the Lamp keypad function Shift 1 When the lamp is turned off the absorbance screen displays the words Lamp off and the lamp icon appears with an X through it Use the Lamp key Shift 1 to shut the lamp off or turn the lamp on manually display the number of times the lamp has ignited
52. 1 Contacting Waters technical service e eeeeeeee eene ee eee eene eee eee eene eene 4 2 Maintenance consideratiOns eee eor eo eio eee eere oeste seen saou o aepo s Tena apo aa penna Ru 4 2 naut and eebe Eegeregie 4 3 Bsp EP ais cibos ite istorbte fuse DH pee pe Cen RD Hed eat AE ERES 4 3 Proper operating procedures cccccccccccccecccccccccccecccccceccceccceccsccceccceecscececeess 4 4 Removing the front left panel eer 4 4 Routine migintenabQe sie rop PERI Eonar Ieee EUM RENTA EE ond EX RUF aC VR ANNENS 4 5 Maintaining the flow Gell eene riri eoe eren egen eEkEEKRE ERC esr caasas 4 5 RE ini the ilow E MR I MT 4 6 Removing and cleaning the flow ce 4 7 Disassembling and reassembling the flow cell 4 8 Replacing the lamp eei rn tr rarae enne no gae ini es desessdseeasecessavasssgasacseis eases 4 16 Beomcung E 4 18 Installing the new lamp e eeeeeeeeeeeeeeenn nnn nnn n nennen nnne nhan nnns 4 21 Recording the new lamp serial number REENEN 4 23 Seting the lamp threshold edere re E pU ETE a CREE UH RAS 4 24 Table of Contents xi Replacing fuses a oer REESE ERES eege dee 4 25 5 Error Messages Diagnostic Tests and Troubleshooting 5 1 ExrroranessadPes iiis ah a PRESE NES SIS TAN AEON E EEEE OS EEEE ESEE 5 2 EE 5 2 Error messages preventing Operation 5 5 User selected diagnostic tests ciscicisssesscdscssissessssacdasescdssnsaasssccasdsdsasssanescenscoeasss 5 9 OE 5 9 Using the diagnostic LeBb
53. 2 to 60 seconds The figure below shows the difference between a single pulse and a rectangular wave Setting the pulse period or signal width using SW1 or SW2 n Seconds lt lt e Single pulse n Seconds s Rectangular wave Setting display contrast The Contrast function allows you to adjust the contrast of the detector display screen When you press the Contrast key Shift 6 the Display Contrast screen appears Preparing the Detector Display Contrast screen Use the A and YF keys to adjust display contrast Use the s and t keys to adjust the contrast of the display Displaying system info The System Info key Shift 4 displays information about the detector including the serial number and the firmware version number Tip Use the scroll bar to see all of the message The Firmware Rev and ChkSm values shown here are examples only They do not indicate the released version s information System info screen example Waters 2489 FE pd an tart of 2009 Waters Corp E a of message ChkSm se44TcO4b Firmware Heu 1 40 1981 Jan 05 2009 H S203 Waters Corp Bl End of message ChkSm we44To04b S SM BOBSTOISN P Using Help The detector has limited context sensitive help When you press Shift HOME from a point in the program that has a Help screen associated with it the appropriate screen appears Using the operator int
54. 20 unpacking and inspecting 2 5 warming up 3 28 deuterium lamp installing 4 22 optics 1 4 replacement 4 16 DIAG key 3 10 diagnostic tests auto zero offset 5 11 5 13 change lamp 5 11 5 16 contact closures and events 5 11 DIAG key 3 10 display test 5 11 failure 3 3 5 2 fix set absorbance 5 11 fix set voltage 5 11 generating test peaks 5 11 5 17 input and output 5 11 5 12 keypad test 5 11 5 17 lamp display and keypad 5 11 5 15 optical filter override 5 11 procedure 5 9 5 19 reset 5 10 sample and reference energy 5 10 5 11 Index 4 service 5 11 5 19 setting a fixed absorbance value 5 13 setting a fixed voltage output 5 14 startup 3 2 sticky 3 6 5 9 test display 5 17 user selected 5 9 5 19 using 5 1 5 22 verification failure 3 28 difference plot 3 16 diffraction grating 1 4 dimensions B 4 dirty flow cell 4 5 disabling external events 3 13 inputs 3 13 disassembling the flow cell 4 8 4 16 display absorbance 3 4 absorbance trace 3 10 diagnostic test 5 11 5 15 lamp use statistics 3 11 options 3 10 system information 3 25 test 5 17 display diagnostic test 5 17 drift specification B 2 dual wavelength mode changing to single 3 32 description 1 11 3 9 functions 3 31 key 3 9 operating in 3 31 3 34 parameters 1 12 3 15 E EC Authorized Representative vii electrical connections 2 9 2 10 specifications B 3 electrical symbols A 12 Empower controlling the detector using 2 14 enabling ch
55. 4 0000 AU For convenience you can also specify sensitivity User selected diagnostic tests 5 13 AUFS using this diagnostic test The allowable AUFS range is from 0 0001 to 44 0000 AUFS Fix absorbance diagnostic display AP Fiz Absorbance 5 40000 fau 0000 aurs E This test sets the voltages on the analog output channels based on the current AUFS setting This is a sticky diagnostic test Setting fixed voltage output From the Input amp Output choice list press 3 Fix voltage to select a voltage for the analog output You can select a voltage for both output channels in the 0 10 V to 2 10 V range Fix Voltage diagnostic display OFF fv ona The voltage is driven on the selected analog channel A or B This is a sticky diagnostic 5 14 Error Messages Diagnostic Tests and Troubleshooting To monitor contact closures and setting switches 1 From the Input amp Output choice list press 4 Contact closures amp events to monitor the four contact closure inputs and to control the two switch outputs Contact closures amp events diagnostic display Co Chat mark 0 Lamp swi ES C AMO zero ow Ra C Inject al The Input amp Output diagnostic test allows real time monitoring of the state of the contact closure inputs A solid filled in circle indicates the contact closure is closed ON High An open empty circle indicates the contact closure is open OFF Low
56. 717plus autosampler terminal Pin 1 Inject Start Inject Start any one of three paired with Pin 2 Inject Start Inject Start any one of three paired with Detector inject start connections 717plus connector 2489 connector II Q2 oz Inject Start Qe Inject Start 12 Pom Ojc Ground 11 Start ect Lamp On 10 Pom Ile L Lam On 9 Start Olo L art mark Inject O c 2 Chart mark 1 7 Start Q r L Ground 6 Inject O 2 c Auto Zero Start lol c 0 Auto Zero pent Qi L Q c N Ol L Connecting the detector to a fraction collector The detector can trigger a fraction collector based on these things Timed events see Timed events on page 3 35 Threshold levels see Threshold events on page 3 37 You can connect the fraction collector to one of the detector s two programmable switches SW1 or SW2 and program the timed event threshold or ratio at the front panel Connecting to other devices 2 33 You can also connect the fraction collector to trigger a chart mark event input each time a tube is changed at the fraction collector The table below indicates the detector to fraction collector and autoinjector to fraction collector connections Detector connections to the fraction collector 2489 connection Fraction collector I Pin 3 Ground Pin 1 Detector In II Pin 6 Chart mark Pin 10 Event
57. A 3 C Calibrate key 3 11 3 30 4 17 calibration algorithm 1 9 errors during startup 3 30 manual 3 11 3 30 Cancel key 3 11 caution symbol A 5 CE key 3 12 Change Lamp diagnostic test 5 11 5 16 changing channels 3 10 contrast 3 11 filter time constant 3 13 Index 2 modes 3 32 scale on an absorbance trace 3 10 time constant 3 13 wavelength from dual to single 3 9 3 32 wavelength from single to dual 3 9 3 32 channel changing 3 10 I and II outputs 2 9 on 3 5 selector 3 5 chart mark configuring event inputs 3 23 generating 2 27 3 9 generating from the 2695 separations module 2 18 timed event parameter 3 35 Waters 600 Series Pump connections 2 30 Chart Mark key 3 9 chart polarity function 3 13 parameter 3 19 chart recorder connections 2 25 2 27 charting difference plot 3 16 MaxPlot function 3 15 RatioPlot function 3 15 checking peak response 3 29 chemical hazard warning A 5 cleaning the flow cell 4 5 4 16 Clear Field key 3 12 clearing editing changes 3 12 events 3 41 configuration screens 3 22 CONFIGURE key 3 10 3 22 3 23 configuring auto zero event input 3 23 detector 3 10 3 22 event inputs 3 23 lamp signal 2 28 with other systems 1 2 connecting to 2695 separations module 2 17 2 19 chart recorder 2 25 2 2 7 electrical power 2 9 2 10 Empower 2 20 2 23 eSAT IN module 2 20 2 23 external devices 2 10 fraction collector 2 33 HPLC system 2 6 2 8 other equipment 2 19 2 34 other syste
58. A or Canada report malfunctions or other problems to Waters Technical Service 800 252 4752 Otherwise phone the Waters corporate headquarters in Milford Massachusetts USA or contact your local Waters subsidiary Our Web site includes phone numbers and e mail addresses for Waters locations worldwide Go to www waters com When you contact Waters be prepared to provide this information Completed normal operation checklist for the method you are using Nature of the symptom Instrument serial number Flow rate Operating pressure Mobile phase s Detector settings Type and serial number of column s Sample type Control mode Empower MassLynx FractionLynx No interaction or other Software version and serial number For complete information on reporting shipping damages and submitting claims see Waters Licenses Warranties and Support Services Maintenance considerations 4 2 Perform the procedures in this chapter when you discover a problem with a 2489 detector component or during preventive maintenance Maintaining the Detector Safety and handling Observe these warning and caution advisories when you perform maintenance operations on your 2489 detector Warning To prevent injury always observe Good Laboratory Practices when you handle solvents change tubing or operate the 2489 detector Know the physical and chemical properties of the solvents you use See the Material Safety Data Sheets for the sol
59. Always flush and purge the flow cell as your initial attempt to correct these problems You should flush the flow cell whenever noise is higher than expected Noise test results are not meeting specifications the detector fails to normalize Caution To avoid damaging the flow cell during reverse flushing do not overpressure the cell If you use buffered mobile phase flush it from the detector before powering off Caution fthe flow cell will not be used for several days flush it with the clean mobile phase such as a water acetonitrile or water methanol then cap the flow ports or dry the flow cell with pure nitrogen or pure helium To prevent flow cell failure do not connect any tubing or device that can create backpressure exceeding the flow cell s maximum rating of 3447 kPa 34 bar 500 psi Requirement Always use well degassed eluents To flush the flow cell 1 Stop the solvent flow and remove the column 2 Replace the column with a union or piece of tubing Caution If the mobile phase is not compatible with water flush with an intermediate solvent first 3 Flush the detector with HPLC quality water Maintaining the Detector Pump 100 methanol through the flow cell to clean it internally Do not exceed 3447 kPa 34 bar 500 psi Pump a strong cleaning solvent such as isopropanol through the flow cell optional Do not exceed 3447 kPa 34 bar 500 psi Caution If the mobile phase is no
60. ER TERREA PULS SRA BJ CRIT RE x8 EE SUUS R e TEI BASEL EHE IK I o SB CO A It Pe EAS HH YP AS BERE AE Sie JRE Be PSEA RUE TRE BJ RES E ITA r TY fg A APER RENE UA gate Rma E KOK BARE BW Hs He 7J o au IS Sell amp 9sg FAS a3 ole FANA AG Ball FS SAAN SA Ss eras sch i 249 828 9c DAA ASHA ee cj 2 LE DO SSE ASHI OMA Hlas Nonmetallic FSS El amp eto e 3 et Tetrahydrofuran THF z E SN UIS SA ASotA NAS 8 st OS el Methylene D 8 C O E amp A amp Dimethyl sulfoxide Was S35 Eq SO re tas AA SAAB F UCVS RANA Ee SUV Fa TER La ES LC SL JES NERY v Fa FORIE CIS mor JLT KAY r4 IC db 5 Jed LCS Su X LSCZJ LI EMH Bok F aT re LZRVY CK FAV HEEF TZI FRIE FAV FATHA 8j OBE KIMBER ea SAVIO K AVY e HEA FLY ROVAF UV AVAKY Fit HEEF TOMES El TRAD HY OBA Fa TiO CVE CREER L3 Warnings that apply to all Waters instruments A 9 Warning The user shall be made aware that if the equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impaired Attention L utilisateur doit tre inform que si le mat riel est utilis d une facon non sp cifi e par le fabricant la protection assur e par le mat riel risque d tre d fe
61. Installing the Detector acer dip rein entes bt PbRR Uo o duae donde 2 1 Preparing for installation 22 55 9 2 p paEISe tet epELIIIEEIIdo pas eb cR 2a Maca ies e PAR D EMI isisa 2 2 Site selection and power requirements eese eene nennen enean nnn 2 3 BUB se lechon uoa icon ideo Gabe Hilt EA eM ea M Ei paid 2 3 EE 2 4 Unpacking and inspecting sooo n e WE Ar EHE E PUE FUR T 2 5 Making plumbing connections 432 a reo p ritos teo ERR rb pux eS EE UE Ro ovp e o IE PE URP PKUu 2 6 Making electrical power connections eee ee eee enne enne nn nena nennen noon 2 9 Waking signal connocttofig usos ie Rie PR HIR Sirsa MU EUN D MR UM MEME 2 10 Making Ethernet connections cesses eene eene ene enmt nnn 2 13 Connecting to other devices c eeeeeee eere esee eee eee eee eee ette eee ettet ette tete een 2 19 Connecting the detector to Empower using an e SAT IN module 2 20 Connecting the detector to a 745 745B 746 data module 2 23 Connecting the detector to a chart recorder ssssessssssssesssrsseereresreerrererrere 2 25 Connecting the detector to the Waters 600 series pump 2 28 Connecting the detector to the Waters 717plus autosampler 2 31 Connecting the detector to a fraction collector sss 2 33 4 Preparing the Det gbOE 5 m a ro FERFETRTEQREE EISE T EPEOUERRIRIFEEFEEPETETE SES EEIOS Ves 3 1
62. Maintaining the Detector Disassembling the flow cell Caution To prevent contamination use powder free finger cots or gloves when disassembling inspecting cleaning or replacing parts within the Waters TaperSlit Flow Cell or when removing or replacing the flow cell within its assembly The TaperSht flow cell consists of these components Flow cell body Cuvette lens Split ring cuvette lens holder Cuvette lens screw Lens mount screw Exit window Exit window mount Entrance lens holder Entrance lens e Entrance lens mount Two gaskets Waters TaperSlit flow cell Cell entrance P di For replacement parts for the TaperSlit flow cell use the Flow Cell Rebuild Kit Maintaining the flow cell 4 11 Tip Use nitrogen to clean the flow cell Use ethanol or methanol to clean the lenses and window To remove the individual parts of the flow cell for cleaning or replacement 1 With the notches on the flow cell cuvette lens end facing you use the flat blade screwdriver or a coin to remove the cuvette lens screw Insert the cotton end of the lint free swab into the cuvette end of the flow cell assembly and remove the split ring and the cuvette lens Using the screwdriver remove the exit lens mount screw Using plastic tweezers pull out the exit window from inside the exit lens mount screw Using the plastic tweezers or the lint free swab remove the clear plastic gasket Flip the flow c
63. Obtaining a MaxPlot The detector obtains a MaxPlot by monitoring absorbance at two selected wavelengths while plotting the maximum absorbance for each sample component To run a scan using the MaxPlot function 1 Besure the detector is operating in dual wavelength mode see page 3 32 From the absorbance screen press Next to reach screen 3 of 5 In the Data out field press 5 MaxPlot A B Press Enter to select the MaxPlot function Or om 8 ge Return to the absorbance screen by pressing the HOME key Programming timed events threshold events and methods You can retrieve as many as five methods which the detector references as numbers 1 to 5 An asterisk in the method number icon see the table on page 3 5 tells you the current conditions are not stored If you are using a stored method the method number appears on the absorbance screen If you edit a parameter such as wavelength or AUFS you are editing the current conditions Method which can be stored as a method You can store the method in one of the 10 available method storage slots or you can replace the current method with one of the methods previously stored When you recall a previously stored method you replace the existing method conditions with those of the stored method The method number displayed in the absorbance screen is that of the retrieved method until you make a change Any parameter change for example wavelength or AUFS alters the current co
64. R seirce a EO RE SENES 5 11 orsi apros LEBER eege eege 5 19 oT oubleshootbhg 2 3 2 5 rte iea itasse secus EE E AA EN ivi 5 20 When vod CODD E 5 20 EE T 5 20 Foner MPO toa gcd dd EE 5 21 Hardware troubleshooting oo die Ei rH REPE CHO RC UO RE Dea ESAE Ct em 5 21 WT TE A 1 Marnins Sy E A 2 KN A 2 PSC A aired oo dati arias E ei A 3 Caution E TE A 5 Warnings that apply to all Waters instruments cccccccccccscccecesseeseesecs A 5 Electrical and handling symbols ERR ee eene eere nan A 12 EE A 12 eet EE EE EE A 13 B Specifications uondeke bebe peo eap Era UR iR e E FE V bid KR DRESS B 1 Operational spectfICabtlOnS 2 coo oo eorr p eror eor o Vaso navarro as s r eb RE Tana B 2 Optical speciDeat JOBS 202a dpi Fia cbav ba o nadkui cdd DIa winners B 4 Optional Waters TaperSlit flow cell specifications e B 5 xii Table of Contents C Solvent Considerations ccccccscssccssccsssscccsccccsccccsccccsccssccesesccsesees C 1 IntroducUORB E C 2 PIECE con tanina Oa C 2 E icine Hnti E C 2 Solvent quality oasis picis Coda eno ea avc de b neater Een ER ern VOR Ga C 2 Preparglion DB EHE un uto oe EP ERA ai C 2 TO eMe Ted C 3 Ban EE C 3 tere d en moa Ea pH RO uU DIRE UU bm RUD S UM M E C 3 Solvent miscibility M C 3 How to use miscibility numbers eese een nemen nnn nnne nnns C 5 Buffered Solv
65. Specify the day of the month the lamp is installed and then press Enter to enter it and move to the next field which specifies the year 8 Specify the year last two digits only and press Enter to update the year and to select the Hours field Tip The Hours field is optional If you are using a lamp for which hours of use are already recorded enter those hours of use If this is a new lamp enter 0 hours are 0 9 Press the HOME key 10 At the OK to store message press Enter to store the serial number and date of installation or press Cancel to cancel the entry 11 When the confirmation message appears press Enter 12 Perform a manual wavelength calibration see page 3 30 Requirement To run the verification procedures with the new lamp installed recalibrate the detector or cycle power to the detector after replacing the lamp Setting the lamp threshold You can set an alarm threshold for the lamp When the number of hours meets or surpasses the threshold an alarm message appears The default alarm threshold is 2000 hours 4 24 Maintaining the Detector The alarm message also appears when you first power on the instrument The lamp threshold screen displays the total hours of lamp use since the lamp s installation Lamp alarm threshold screen Lamp Threshold Alarm Threshold FOOO hours Total since installed 1966 h 00 Replacing fuses The fuse holder is located on the rear panel of
66. Waters 2489 UV Visible Detector Operator s Guide 71500142102 Revision B Waters THE SCIENCE OF WHAT S POSSIBLE Copyright Waters Corporation 2007 2009 All rights reserved Copyright notice 2007 2009 WATERS CORPORATION PRINTED IN THE UNITED STATES OF AMERICA AND IN IRELAND ALL RIGHTS RESERVED THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER The information in this document is subject to change without notice and should not be construed as a commitment by Waters Corporation Waters Corporation assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Waters Corporation be liable for incidental or consequential damages in connection with or arising from its use Trademarks PIC and Waters are registered trademarks of Waters Corporation and Empower e SAT IN FractionLynx MassLynx TaperSlit and THE SCIENCE OF WHAT S POSSIBLE are trademarks of Waters Corporation Tygon is a registered trademark of Saint Gobain Ceramics amp Plastics Inc Other registered trademarks or trademarks are the sole property of their owners Customer comments Waters Technical Communications department invites you to tell us of any errors you encounter in this document or to suggest ideas for otherwise improving it Please help us b
67. al M number The first number always lower than 16 indicates the degree of miscibility with highly lipophilic solvents The second number applies to the opposite end of the scale A large difference between these two numbers indicates a limited range of miscibility For example some fluorocarbons are immiscible with all the standard solvents and have M numbers of 0 32 Two liquids with dual M numbers are usually miscible with each other A liquid is classified in the M number system by testing for miscibility with a sequence of standard solvents A correction term of 15 units is then either added or subtracted from the cutoff point for miscibility Buffered solvents When using a buffer use a good quality reagent and filter it through a 0 45 um filter Do not leave the buffer stored in the system after use Flush all fluid paths with HPLC quality water before shutting the system down and leave distilled water in the system flush with 90 HPLC quality water 10 methanol for shutdowns scheduled to be more than one day Use a minimum of 15 mL for sparge equipped units and a minimum of 45 mL for in line vacuum degasser equipped units Head height Position the solvent reservoirs at a level above the HPLC equipment or on top of the pump or detector with adequate spill protection Solvent viscosity Generally viscosity is not important when you are operating with a single solvent or under low pressure However wh
68. al screens All parameters appear on the first New scan screen for both a zero and a sample scan You can return to screen 1 to review the parameters for either scan type by pressing Next from the Run screen screen 3 of 3 for sample scan or 4 of 4 for zero scan Tip You can press Run from any New scan screen 3 46 Preparing the Detector For both zero and sample scans the table below provides the defaults and ranges for all parameters Sample and Zero scan parameters Parameter Screen Scan type Units Range or default Type 1 Sample and n a Sample scan 1 Zero Zero scan 2 Default 1 range 2 Zero scan only nm Range 190 to 700 nm Default 190 to 700 nm Pace 2 Zero scan only nm min Range 30 to 1000 nm min Default 100 nm min AUFS 2or 3 Sample and AU Range 0 0001 to 4 0000 Zero Default Last number entered Tick mark 20r3 Sample and nm Range 10 to 100 Mark each Zero Default Last number nm entered Zero scan To program a zero scan 1 Press SCAN then 1 New scan and press 2 Zero scan Press Next to advance to the second Zero scan parameter screen Enter the ending wavelength for the zero scan and then press Enter 2 3 Enter the starting wavelength for the zero scan and then press Enter 4 5 Enter a value in the Pace field for the rate at which the detector scans the specified wavelength range Tips The default is 100 nm min The allowable range is from 30 to 1000 n
69. alibration and operating error messages descriptions and recommended actions you can take to correct the problem Startup calibration and operating error messages Error Message Calibration differs n nm Description At startup the unit performs a complete verification which includes remeasurement of all calibration points New calibration points are compared to stored information from the most recent manual calibration If any one of the points differs by more than 1 0 nm the detector displays this message Corrective Action 1 Cycle power to the detector 2 Perform manual calibration 3 Contact Waters Technical Service Calibration not found Stored calibration data not valid Perform manual calibration procedure Calibration unsuccessful Peak out of range n nn nm Results of a calibration operation differ by more than 1 0 nm Unit uses previously stored calibration points 1 Remove cuvette 2 Be sure the front left panel door is closed 3 Flush flow cell Lamp external input conflict A timed event or action from the unit s front panel attempts to change the lamp state in conflict with enabled lamp input contact closure 1 Check contact closure status 2 Check timed events 3 Cycle power to the detector Error messages 5 3 5 4 Startup calibration and operating error messages Continued Error Message Lamp failur
70. alytical cell Linearity 596 at 2 5 AU propylparaben at 257 nm Drift 1 0 x 10 4 AU hr max AT 2 C hr Thermal drift 1 0 x 104 AU C max AT 2 C hr Sensitivity setting range 0 0001 to 4 0000 AUFS Filter setting range Single wavelength 0 1 to 5 0 seconds Dual wavelength 1 to 50 seconds time constant 0 0 to 5 0 second Hamming filter Digital data rates 1 2 5 10 20 40 80 Hz single channel 1 2 Hz dual channel B 2 Specifications Operational specifications Continued Condition Specification Analog output 10 20 40 80 Hz channel A data rates 10 Hz only channel B single mode Optical Component Sp ecifications Lamp source 30 W High Brightness Deuterium lamp 0 5 nm aperture pre aligned 2000 hour warranty front accessible Photodiodes 2 silicon photodiodes matched pair Second order filter Automatic for wavelengths 370nm Wavelength Erbium filter used at startup or on demand calibration filter Flow cell TaperSlit flow cell design Nitrogen purge Purge fitting present on optics bench Path length 10 mm standard analytical Cell volume 10 uL standard analytical Pressure limit 6895 kPa 69 bar 1000 psi Materials 316 stainless steel fused silica Tygon Environmental Specifications Operating 4 to 40 C 39 to 104 F temperature Operating humidity 20 to 9596
71. ameter to any number from 1 to 60 You can use the Scale function to zoom in on a particular section of the trace To display the Scale parameters after you press the Scale key 1 Press Next to display T2 starting time The default is 0 2 Press Next again to display AUI starting or low absorbance The default is Auto 9 Press Next again to display AU2 ending or high absorbance The default is Auto By entering appropriate times and absorbance numbers in the four scaling parameter boxes you can zoom in on one section of the current absorbance trace For AUI and AU2 press CE to reset to auto TI represents the left hand side of the trace or ending time to be displayed The default is 30 T2 represents the right hand side of the trace or starting time The default is 0 3 20 Preparing the Detector The figure below shows a 60 minute trace of continuous injections of caffeine and propyl paraben in 1 1 methanol water Scaled trace of continuous injections with T1 changed to 60 D Ka T rd min 40 The figure below shows a five minute scaled trace or zoom of the 60 minutes of continuous injections shown in the previous figure T1 is changed to 5 T2 is changed to 0 AU1 and AU2 remain as auto Scaled trace for 5 minutes of continuous injections with T1 changed to 5 A Ko r a m J 1 ka min The figure below shows a 60 mi
72. ance of a selected channel s analog output 2 V connector scaled to the AUFS setting for that channel and adjusts the absorbance value according to the voltage and absorbance offsets for that channel Example For the 2 V output with 0 used for both the voltage and absorbance offsets Volts out Absorbance x 2 V AUFS 3 14 Preparing the Detector Detector functions Continued Function Description Analog out In addition to the selections for single X above you dual A can chart the same parameters on the other channel at a different wavelength and you can chart the following parameters MaxPlot Charts the absorbance of multiple compounds with different absorbances at two different wavelengths on a single data channel Scaling for MaxPlot is the same as for Absorbance above except that the charted absorbance is the larger of the two absorbances as measured on channel A and channel B The detector uses the AUFS absorbance offset and voltage offset of the selected channel regardless of which absorbance channel A or channel B is larger Volts out Larger absorbance A or B x 2 V AUFS of selected channel RatioPlot A B Charts the ratio of absorbances at two wavelengths Theoretically the ratio is constant for a pure chromatographic peak and variable for an impure peak This mode applies the three ratio parameters on screen 5 of 5 see the figure below Minimum AU This setting de
73. ar all desired methods When you press HOME the absorbance screen s method number icon displays an asterisk Clearing events You can clear only timed or threshold events without resetting any other operating parameters To clear all active timed or threshold events 1 2 Return to the Method choice list by pressing METHOD Shift A B Press 6 Clear events Using the operator interface 3 41 Result A message appears asking whether you approve of clearing all active events Tip If you press Enter All timed and threshold events in the method are cleared All other operating parameters of the method A AUFS etc are unaffected If you press Cancel Shift 0 the Method choice list appears When you press HOME the absorbance screen s method number icon displays an asterisk Scanning spectra The detector must make two scans to produce an absorbance spectrum Zero scan A reference scan that characterizes the absorbance spectrum of the solvent in the cuvette or the flow cell Sample scan An absorbance scan of the analyte in solvent after subtracting out the zero scan of the solvent to provide the actual spectrum of the sample The detector can measure the spectrum of a sample using the cuvette or the flow cell See page 3 56 and page 3 59 for scanning procedures Rule When using the cuvette if the contents of the flow cell change you must rerun the zero scan Before you begin Befor
74. art mark event inputs 3 23 external events 3 13 inputs 3 13 ending wavelength 3 42 Enter key 3 12 entering negative numbers 3 12 entrance slit 1 4 environmental specifications 2 3 B 3 equipment guidelines v A 5 Erbium scan 3 44 error messages 5 1 5 22 errors calibration 3 30 fatal 5 5 startup 3 30 5 2 eSAT IN module connecting to 2 20 2 23 Ethernet connections 2 13 2 14 default address 3 22 interface 2 13 2 14 event inputs auto zero 3 23 chart mark 3 23 configuring 3 23 default 3 23 functions 2 10 inject start 3 23 lamp 3 24 external events disabling 3 13 enabling 3 13 F failure of startup diagnostic tests 3 3 features MaxPlot function 1 14 RatioPlot function 1 14 spectral 1 9 filter time constant changing 3 13 filter time constant function 3 16 parameter 3 19 filters filter setting specification B 2 noise 1 7 optical override 5 11 replacing solvent reservoir 4 5 second order 1 3 1 4 1 10 time constant 3 16 fix set absorbance diagnostic test 5 11 fix set voltage diagnostic test 5 11 flammable solvents A 3 flow cell cleaning 4 5 4 16 conditions 3 57 contamination 3 28 damaged 4 13 4 15 description 1 6 1 8 dirty 4 5 disassembling 4 8 4 16 exploded view 4 13 flushing 4 6 frontal view 4 11 gasket 4 13 head on view 4 11 inspecting 4 5 4 16 lens 4 13 optics 1 4 optional 4 15 reassembling 4 8 4 16 Index 5 rebuild kit 4 11 rebuilding 4 14 4 15 removing 4 7 replacing 4 15 4
75. calibrating mass spectrometers consult the calibration section of the operator s guide for the instrument you are calibrating In cases where an overview and maintenance guide not operator s guide accompanies the instrument consult the instrument s online Help system for calibration instructions Quality control Routinely run three QC samples that represent subnormal normal and above normal levels of a compound Ensure that QC sample results fall within an acceptable range and evaluate precision from day to day and run to run Data collected when QC samples are out of range might not be valid Do not report these data until you are certain that the instrument performs satisfactorily ISM classification ISM Classification ISM Group 1 Class B vi This classification has been assigned in accordance with CISPR 11 Industrial Scientific and Medical ISM instruments requirements Group 1 products apply to intentionally generated and or used conductively coupled radio frequency energy that is necessary for the internal functioning of the equipment Class B products are suitable for use in both commercial and residential locations and can be directly connected to a low voltage power supply network EC Authorized Representative Waters Corporation Micromass UK Ltd Floats Road Wythenshawe Manchester M23 9LZ United Kingdom Telephone 44 161 946 2400 Fax 44 161 946 2480 Contact Quality manager vii viii
76. connection as shown in the following figures 2 26 Installing the Detector Chart recorder 2 V output connections on the detector 2489 connector Analog 1 Analog 1 Ground Analog 2 Analog 2 Switch 1 Switch 1 Ground Switch 2 0 Switch 2 Chart recorder OO OO JO Om PS GO A 1 Analog 1 2 Analog 1 3 Ground 4 Analog2 5 Analog2 6 Switch 1 7 8 9 1 Switch 1 Ground Switch 2 O Switch 2 Chart recorder Chart marks You can generate a chart mark from the front panel of the detector A chart mark signal results from one of these actions or events You press the Chart mark key on the detector keypad e You program a timed event to generate a chart mark Signal is received from one of the chart mark inputs on the analog connector Connecting to other devices 2 27 Connecting the detector to the Waters 600 series pump 2 28 To connect the detector to the pump locate the detector in a position that satisfies the site requirements on page 2 3 Plumbing connections Make line connections as described in Making plumbing connections on page 2 6 Lamp on off connections In addition to making the connections described below you must configure the lamp on off signal at the front panel You must change the default from Ignore to High or Low See the discussion page 3 23 for more information Using sig
77. cts of temperature Temperature affects the solubility of gases If the heat of solution is exothermic the solubility of the gas decreases when you heat the solvent If Mobile phase solvent degassing C 7 the heat of solution is endothermic the solubility increases when you heat the solvent For example the solubility of He in H O decreases with an increase in temperature but the solubility of He in benzene increases with an increase in temperature Effects of partial pressure The mass of gas dissolved in a given volume of solvent is proportional to the partial pressure of the gas in the vapor phase of the solvent If you decrease the partial pressure of the gas the amount of that gas in solution also decreases Solvent degassing methods This section describes the solvent degassing techniques that will help you to attain a stable baseline Degassing your solvent also improves reproducibility and pump performance You can use either of the following methods to degas solvents Sparging with helium Vacuum degassing Sparging Sparging removes gases from solution by displacing dissolved gases in the solvent with a less soluble gas usually helium Well sparged solvent improves pump performance Helium sparging brings the solvent to a state of equilibrium which may be maintained by slow sparging or by keeping a blanket of helium over the solvent Blanketing inhibits reabsorption of atmospheric gases Sparging can change
78. ctueuses Vorsicht Der Benutzer wird darauf aufmerksam gemacht dass bei unsachgem Der Verwenddung des Ger tes die eingebauten Sicherheitseinrichtungen unter Umst nden nicht ordnungsgem funktionieren Attenzione si rende noto all utente che l eventuale utilizzo dell apparecchiatura secondo modalit non previste dal produttore pu compromettere la protezione offerta dall apparecchiatura Advertencia el usuario deber saber que si el equipo se utiliza de forma distinta a la especificada por el fabricante las medidas de protecci n del equipo podr an ser insuficientes SEE EHA ZR TE RE TG A8 UL i fl 0 r 8 ERE 2 AREE P P e D HI CREE TS BCE SIS BR HAAMER A AER te TI E TE EA ABA THe BE EIER GAN AD MASAMI SASHA 98 8 gr leg suis ASS SS Sul MSoteE PS OO MOS SSoA FS UAE SS ASIAH DCH IA AHO SLICE BSG APL cl LU ESN CORY TIE Chee eA T OL Bases tect L CWS tRaEDS BECA 2 REED OO LICE UT FAY A 10 Safety Advisories Warning To protect against fire replace fuses with those of the type and rating printed on panels adjacent to instrument fuse covers Attention pour viter tout risque d incendie remplacez toujours les fusibles par d autres du type et de la puissance indiqu s sur le panneau proximit du couvercle de la boite fusible de l instrument Vorsicht Zum Schutz gegen Feuer die Sicherungen
79. d 2 of 5 3 of 5 and so on see page 3 18 Setting up a run Once you press HOME causing the absorbance screen to reappear and you select a wavelength mode A or 21 you can set up the detector for a run In Using the operator interface 3 13 addition to wavelength mode you must program the following parameters before beginning a run Operating wavelength Sensitivity time constant Analog output sensitivity Depending on other functions that you can perform during a run several other parameters can require programming See page 3 14 and the table on page 3 18 for the function descriptions fields screen number type of function display units allowable ranges and the default settings for the absorbance screen and the secondary function screens Primary and secondary functions You can access the following functions directly from the absorbance screen or by pressing the Next key on that screen Detector functions Function Description Wavelength Defines the operating wavelength for the channel AUFS absorbance Defines the relationship between the absorbance and units full scale the output voltage The output voltage reaches full scale 2 V when the current absorbance attains the AUFS value Caution Changing the sensitivity AUFS setting affects the 2 V output Analog out You can chart the analog connections for the following single i parameters Absorbance Charts the current absorb
80. d Inject 1 Hold Inject 2 Hold Inject 2 Ground Chart Out Chart Out A DO OD rd OO PS GO prm SS 2489 connector II 1 2 3 4 5 6 7 8 9 1 0 Inject Start Inject Start Ground Lamp On Lamp On Chart mark Chart mark Ground Auto Zero Auto Zero Note If the injector is an e2695 separations module running in Ethernet mode or a 2707 autosampler the inject start cable should not be connected However if the injector 1s an e2695 separations module running in IEEE mode the inject start cable should be connected Making electrical power connections 2 15 2 16 Turning the detector lamp on or off Before you can turn the detector lamp on or off from the separations module you need to configure the lamp on off signal at the front panel You must change the default lamp configuration parameter setting from Ignore to High or Low See the discussion Configuring event inputs contact closures on page 3 23 for more information After configuring the detector lamp on off signal you can turn the lamp on or off from the separations module by making the connections shown in the table and figure below Detector connections to separation module lamp on or off 2695 separations module A outputs 2489 detector Il Pin 1 Switch 1 Pin 4 Lamp On Off Pin 2 Switch 1 Pin 5 Lamp On Off Separations module connections to the
81. d channel In single wavelength entry mode you cannot independently control wavelength on channel B Channel selector Changes the channel when you press AP A B The icon of the selected channel overlaps that of the other channel ON Channel On Displays ON A or ON B for the channel a o timed or threshold event is programmed on Channel trace When you press TRACE displays only the channel being viewed Numerical Absorbance Displays current absorbance for the field selected channel tn Lamp on Indicates the lamp is turned on be Lamp off Indicates the lamp is turned off Using the operator interface 3 5 3 6 Absorbance and message screen icons Continued Icon or field Icon field name Function gt Shift off Blank Shift off vu Shift on Indicates the shift is on for one key press Single wavelength Indicates the detector is operating in single channel mode Multiwavelength Indicates the detector is operating in dual channel mode Keypad unlock Indicates unrestricted keypad entry Keypad lock Indicates parameter changes are not allowed The keypad locks when the instrument is under control of an external data system remote mode only Sticky diagnostic on Indicates a sticky diagnostic setting is active See page 5 9 for an explanation of sticky diagnostic settings Local method number Indicates the detector is not controlled by a data system The icon s cursive
82. d performance Two contact closure outputs The detector has two configurable switches each of which can accommodate a maximum of 30 VDC 1 2 A current carrying capacity and 0 5 A current switching The switches SW1 and SW2 can trigger fraction collectors and other external devices as well as activate according to time absorbance threshold or ratio criteria Therma wander management To mitigate thermal instability caused by ambient temperature changes the detector s insulation ensure air flow across the optics bench and a variable speed fan that runs at higher or lower speeds as needed Median Baseline Filter MBF A variation of the data mode the MBF decreases the effects of gradient separations on the chromatographic baseline It enhances the UV detector s baseline stability by decreasing its curvature making the development of integration methods easier Detector description 1 3 Principles of operation To use the detector effectively become familiar with its optical and electronic design and the theory and principles of its operation This section describes the following parts and functions of the detector Optics Wavelength verification and test Flow cell Electronics Detector optics The Waters 2489 UV Visible detector optics are based on a Fastie Ebert monochromator and include these components High brightness deuterium D lamp Two mirrors one off axis ellipsoidal mirror and one spher
83. detector for turning the lamp on or off 2695 connector A 2489 connector II 1 Inject Start Switch 1 1 2 Inject Start Switch 1 2 3 Ground Ground 3 4 Lamp On Switch 2 4 e 5 Lamp On Switch 2 5 A 6 Chart mark Switch 3 6 A 7 Chart mark Switch 3 7 P 8 Ground Switch 4 8 2 9 Auto Zero Switch 4 9 2 10 Auto Zero Ground 10 Run Stopped 11 2 Run Stopped 12 2 Installing the Detector Connecting the detector to a separations module You can connect the detector to the separations module when the detector is not under the control of the Empower software to perform the following functions e Auto Zero Chart mark on Inject Generating auto zero To generate the Auto Zero function of the detector at the start of an injection make the connections summarized in the table and illustrated in the figure the follows it Detector connections to a separation module to generate auto zero 2695 separations module B inputs and outputs Pin 1 Inject Start Pin 9 Auto Zero Pin 2 Inject Start Pin 10 Auto Zero 2489 detector Il Before you can generate an auto zero from the separations module you need to configure the auto zero signal at the detector s front panel The default auto zero signal is Low See the discussion Configuring event inputs contact closures on page 3 23 for more information Making electrical power connections 2 17 2 18 Separations
84. detector s rear panel also includes one Ethernet interface connector for digital signal communications Use this connector for devices such as these Network adapter card in the Empower workstation Solvent manager MassLynx version 4 1 or higher The Ethernet connector mates with standard Ethernet cable Caution To avoid possible damage to components power off all instruments on the Ethernet connector before you connect an Ethernet cable to an instrument Making Ethernet connections with Waters data systems When controlling the detector from a Waters data system or controller Empower or MassLynx workstation you can use the Ethernet interface to send and receive information from the data system Making electrical power connections 2 13 When connecting via the Ethernet to these Waters data systems you should be aware of the following When in dual wavelength mode you must select a data rate of 1 point per second in the data system method editor In Empower software the data rate defaults to 1 point per second The maximum range of the detector time constant setting depends on the wavelength mode and data rate selected See the page 3 18 Empower allows the detector to operate in both single and dual wavelength modes with a wavelength range of 190 to 700 nm and as many as 4 0 AUFS To connect Ethernet cables from the detector to a Waters data system 1 Connect the single receptacle end of the Ethernet cable to y
85. dow holder facing down use the torque wrench to tighten the exit window screw into the flow cell body to 0 904 N m 128 1n oz or 8 1n 1b 4 14 Maintaining the Detector Tip To ensure complete compression of the gaskets after replacing the exit window and torquing the exit window screw and after replacing the cuvette lens and torquing the cuvette lens screw you must flip the flow cell body and torque the entrance lens screw to 0 904 N m 128 in oz or 8 in Ib a second time 12 Flip the flow cell body again and torque the entrance lens screw to 0 904 N m 128 1n oz or 8 in 1b 13 Flip the flow cell body again to the cuvette lens end and torque the exit lens screw again to 0 904 N m 128 in oz or 8 in lb 14 With the curved side of the cuvette lens facing up replace the lens in its screw mount 15 With gloved fingers place the split ring onto the cuvette lens and press it firmly until it 1s flush on all sides 16 Using the cuvette tool turn the cuvette lens screw into the exit lens mount end of the flow cell body 17 Torque the cuvette lens screw to 0 904 N m 128 in oz or 8 in lb 18 Follow the procedures in the next section to refit the flow cell in the flow cell assembly Replacing the flow cell Caution To prevent contamination use powder free finger cots or gloves when disassembling inspecting cleaning or replacing parts within the Waters TaperSlit Flow Cell or when removing or replacing t
86. e Description Lamp indicates Off when it should be On Corrective Action 1 2 Check lamp icon Cycle power to the detector 3 Replace lamp Lamp lighting failure The lamp failed to ignite 1 Cycle power to the detector Check lamp power connection Replace lamp Peak not found Erbium n nm Erbium filter calibration range does not contain a local maximum Remove cuvette 2 Be sure the front left panel door is closed 3 Flush flow cell Peak not found 656 nm deuterium Unit sensors cannot determine 656 nm peak 1 Remove cuvette 2 Be sure the front left panel door is closed Flush flow cell Error Messages Diagnostic Tests and Troubleshooting Startup calibration and operating error messages Continued Error Message Wavelengths span 370 nm Order filter not in use Description In dual wavelength mode If both selected wavelengths gt 370 nm the detector applies the second order filter to block unwanted UV light If both selected wavelengths lt 370 nm the detector removes the second order filter If the selected wavelengths bracket the 370 nm threshold the detector does not apply the second order filter and issues a warning message that any data collected for wavelengths above 370 nm may contain inaccuracies because of possible UV light interference second order effects
87. e detector 3 2 initiating a scan 3 9 inject signal 3 23 Inject Start Waters 600 Series Pump connections 2 31 Waters 717plus Autosampler connections 2 32 input and output diagnostic tests 5 11 5 12 inputs disabling 3 13 enabling 3 13 signals 2 12 inspecting detector 2 5 flow cells 4 5 installation site selection 2 3 installing a new lamp 4 21 4 23 intended use v interface bus 2 13 2 14 inverting the chart 3 13 ISM classification vi K keypad key 3 12 key 3 9 3 25 e key 3 12 A B key 3 4 3 10 Auto Zero key 3 9 Calibrate key 3 11 3 30 4 17 Cancel key 3 11 CE key 3 12 Chart Mark key 3 9 Clear Field key 3 12 CONFIGURE key 3 10 3 23 Contrast key 3 11 decimal point key 3 12 description 3 9 DIAG key 3 10 diagnostic test 5 11 5 17 Enter key 3 12 functions 3 7 3 9 help key 3 9 3 25 HOME key 3 9 WAX key 3 9 3 31 3 32 Lamp key 3 11 lamp display and keypad diagnostic tests 5 11 5 15 Lock key 3 11 locking 3 11 METHOD key 3 10 3 36 Next key 3 10 numerical keys 3 10 Previous key 3 10 Reset key 3 9 Run Stop key 3 9 Scale key 3 10 3 20 SCAN key 3 9 3 45 Shift key 3 10 System Info key 3 11 TRACE key 3 10 3 20 up down arrow keys 3 9 using 3 7 3 12 keypad lock icon 3 6 keypad unlock icon 3 6 L 11 key 3 31 AAA key 3 9 3 32 laboratory acquisition and control environment LAC E 2 20 lamp change 5 16 Index 7 Change Lamp diagnostic test 5 11 5 16 configuring lamp event inputs 3 24
88. e intensity of the deuterium source lamp varies by wavelength as shown in the figure below 4 16 Maintaining the Detector Deuterium lamp sample beam intensity profile Intensity 190 Wavelength nm 700 Lamp energy and performance As the lamps used in traditional detectors age the signal to noise performance of the instrument degrades Determining the longevity of useful lamp life can be difficult because of varying user requirements and individual lamp behavior The detector s design also compensates for the lamp energy changes that occur across the deuterium spectrum as well as lamp aging Thus the detector can operate at the same high signal to noise performance at longer wavelengths of the visible range without the use of a second lamp such as tungsten The detector performs several self diagnostic tests when powered on or when the Calibrate key is selected One of these tests is the lamp optimization software routine After the detector verifies the calibration of the monochromator it evaluates energy levels in several characteristic regions across the spectrum The integration time of the front end electronics is adjusted to maximize the signal within these regions The effect is to maintain a high signal to noise ratio and operate with a clean signal When operating the detector run the lamp optimization software routine at least once a week Eventually the lamp s signal becomes low enough that replacement becom
89. e of operation for the detector The detector supports monitoring of a single wavelength from 190 nm to 700 nm settable in 1 nm increments on channel A You can configure the analog outputs for channel B while the detector is operating in single wavelength mode so you can use channel B to obtain additional information about the wavelength selected on channel A In single wavelength mode the detector automatically engages the second order filter for wavelengths 370 nm and above and removes it for wavelengths under of 370 nm The second order filter is an optical filter that blocks unwanted ultraviolet UV light from striking the diffraction grating and interfering with absorbance detection above 370 nm You can configure several additional parameters when using the detector in single wavelength mode Primary parameters The following are the values of major parameters that apply to single wavelength mode Wavelength in nanometers Specifies a wavelength for channel A from 190 nm to 700 nm settable in 1 nm increments Sensitivity in AUFS Specifies the scaling factor for the analog output channels and corresponds to the absorbance unit AU value where the analog outputs saturate at full scale values Absorbance units full scale AUFS vary from 0 0001 to 4 000 AU Tip Changing the sensitivity AUFS setting affects the 2 V output Chart polarity or Reverses the polarity of the charted chromatogram Select
90. e to obtain additional information about an analyte by running in the RatioPlot or MaxPlot mode You select any two wavelengths from 190 nm to 700 nm In dual wavelength mode the following conditions apply If both selected wavelengths are greater than 370 nm the detector applies the second order filter to block unwanted UV light If both selected wavelengths are less than or equal to 370 nm the detector removes the second order filter If the selected wavelengths bracket the 370 nm threshold the detector does not apply the second order filter and issues a warning message that any data collected for the wavelength above 370 nm can contain Operational modes 1 11 inaccuracies because of possible UV light interference second order effects Chart out selection modes When operating in dual wavelength mode the detector offers these choices for analog output in addition to the selections offered in single wavelength mode and explained on page 1 10 The default selection for dual wavelength mode is Absorbance Absorbance A and B The standard LC mode where the current absorption is scaled and sent directly out the analog output The scaling depends on the AUFS setting and the absorbance offset The absorbance value is scaled for the 2 V analog output If a setting of 1 AU V is desired you can set an AUFS of 2 0000 for either the A or B output channels that can be controlled independently even in single wavelength mode
91. e you run a spectrum scan specify values for the following parameters 1 Starting wavelength Scanning begins at this wavelength 42 Ending wavelength Scanning ends at this wavelength Pace Rate of scanning in nanometers min Determines how fast the scan is output and data are acquired The scan data are acquired at the 3 42 Preparing the Detector highest possible resolution for the specified pace Specifying a very high pace reduces resolution Pace and sampling resolution examples Pace nm min Sampling resolution nm 100 and less 0 5 200 1 0 400 2 0 The figure below shows two scans of anthracene one overlaying the other At a pace of 1000 nm min the overlaid scan dotted line shows a reduced number of points scanned lowering the resolution relative to the original scan done at a pace of 100 nm min Scan of anthracene at 100 nm min and 1000 nm min 0 5 Pace 100 Pace 1000 Absorbance 0 15 t t 235 250 265 Wavelength nm 190 205 220 Tip The higher the number you enter in the Pace field the lower the resolution of the scan Scanning spectra 3 43 Tick marks This value allows tick marks check marks to be generated at the specified wavelength increment help interpret charted data The figures below show a 190 nm to 600 nm scan of erbium standard in a cuvette at 200 nm min with tick marks specified every 20 nm and the same scan
92. ect Start 2 Inject Start 3 Ground 4 Lamp On 5 Lamp On PRESSURE S 6 Chart mark 7 8 9 1 Chart mark Ground Auto Zero O Auto Zero Connecting to other devices 2 29 2 30 Chart mark connections To make chart mark connections between the detector and pump 1 Using a signal cable make the connections between the detector and the pump that are summarized in the table and illustrated in the figure below 2 Program the pump to provide a pulse output on the selected switch at the beginning of each run See also Waters 600E Multisolvent Delivery System User s Guide Pump and 2489 detector chart mark connections 2489 detector Il 600 series pump terminal Pin 6 Chart mark S1 S2 or S4 Pin 7 Chart mark GND one of four Pump chart mark connections 600 series pump 2489 connector Il CHART PRESSURE a SWITCHES 34 dla s g s Red Q 1 Inject Start 2 Inject Start 3 Ground d Lamp On 5 Lamp On 6 Chart mark 7 8 9 1 CHART m PRESSURE Chart mark Ground Auto Zero O Auto Zero Inject start connections Tip If the detector is connected to an Empower data system use the Inject Start connections to initiate the start of data acquisitio
93. ed on You can program all events on channel A all on channel B or some on channel A and some on channel B Event programming is time based not channel specific 8 Press Next to advance to a new timed event 9 To delete a timed event press CE when the time field is active 10 Press HOME to return to the absorbance screen and then press Run Stop 11 Press Reset Tips If the detector is under the control of the Waters 717plus Autosampler or another external device the Inject Start programmed from that device runs the method If you are working in real time under current conditions method when a power failure or shutdown occurs you lose all timed or threshold events if you do not store them as a method See page 3 39 Threshold events You can program threshold events on channel A and channel B to control the switch contact closure outputs for example when using a fraction collector You can program the switch to change when the programmed output absorbance ratio energy etc on the detector channel A or B is above a Using the operator interface 3 37 specified threshold Below the specified threshold the switch is set as shown in the table Threshold events Set parameters 1 Number Event Set switch 1 2 Set switch 2 Threshold events To parameters Number Set to Below threshold switch state I On Off 2 Off On 3 Pulse Off 4 Rect wave Off
94. ell over to the entrance lens side Using the flat blade screwdriver remove the entrance lens screw Using the plastic tweezers remove the entrance lens from inside the entrance lens screw Using the plastic tweezers or the lint free swab tap and remove the clear plastic gasket 4 12 Maintaining the Detector The following figure shows an exploded view of all parts of the flow cell within the flow cell assembly Waters TaperSlit flow cell exploded view ring Cuvette lens ka Exit window goe holder Gasket m Bef SS body Exit d screw G window 2 GE E ER fiat 6 P an curved Entrance lens screw Inspecting cleaning and replacing damaged flow cell components Caution To prevent contamination use powder free finger cots or gloves when disassembling inspecting cleaning or replacing parts within the Waters TaperSlit Flow Cell or when removing or replacing the flow cell within its assembly Work on a clean flat surface such as a nonparticulating cloth or similar surface To inspect or clean the parts of the flow cell or to replace damaged parts of the flow cell such as the lenses exit window or gaskets follow this procedure and the procedure on page 4 14 Recommendation Replace the clear plastic gaskets each time you inspect and clean the flow cell Maintaining the flow cell 4 13 To inspect and clean the flow cell 1 2 4 Inspect each part of the removed flow cell for di
95. en you are running a gradient the viscosity changes that occur as the solvents are mixed in different proportions can result in pressure changes during the run For example a 1 1 mixture of C 6 Solvent Considerations water and methanol produces twice the pressure of either water or methanol alone If the extent to which the pressure changes will affect the analysis is not known monitor the pressure during the run using the Chart Out terminal Mobile phase solvent degassing Mobile phase difficulties account for 70 or more of all liquid chromatographic problems Using degassed solvents is important especially at wavelengths below 220 nm Degassing provides stable baselines and enhanced sensitivity reproducible retention times for eluting peaks reproducible injection volumes for quantitation stable pump operation Gas solubility Only a finite amount of gas can be dissolved in a given volume of liquid This amount depends on the chemical affinity of the gas for the liquid the temperature of the liquid the pressure applied to the liquid Changes in the composition temperature or pressure of the mobile phase can all lead to outgassing Effects of intermolecular forces Nonpolar gases N O CO He are more soluble in nonpolar solvents than in polar solvents Generally a gas is most soluble in a solvent with intermolecular attractive forces similar to those in the gas like dissolves like Effe
96. ents Mee rc C 6 Head BE E C 6 Solvent E EE C 6 Mobile phase solvent degassing eesssesssesssesssesseessssssesssssssesssssssoossosssoososssoeoso C 7 idm BODIE EE C 7 Solvent degassing methods ccccccesccscccssccsccsscccccccecccecccecccececccceccecccecees C 8 OR hl Ls AMNES I RE eee SP SET ene rena quat toc C 8 Vacnam CE E C 8 Solvent degassing considerations iseiseseecevverekxerekr pisce et ec e oio a C 9 Wavelength selection 4 425 3 iet SEE PER ERES astiassa nas C 9 UV e EE C 10 Breed mobile PRint ie ou ro HS ETDEL F CEDERE CER Ee ELEC CEVER EVER EXE CORR C 10 Wavelength selection for chromophore detect on C 11 Table of Contents xiii xiv Table of Contents Theory and Principles of Operation This chapter summarizes the Waters 2489 UV Visible Detector features and describes the theory and principles of operation Contents Topic Page Detector description 1 2 Principles of operation 1 4 Operational modes 1 10 See also Appendix B for system specifications and Appendix C for information on high performance liquid chromatography HPLC solvent considerations 1 1 Detector description The Waters 2489 UV Visible Detector is a two channel ultraviolet visible UV Vis detector designed for high performance liquid chromatography HPLC applications Waters 2489 UV Visible Detector The detector can operate as a stand alone unit with a chart recorder or integrator or as
97. equires grounded alternating current AC power source minimal power transients and fluctuations a line voltage of 100 to 240 VAC Power consumption is 185 VA 2 4 Installing the Detector operation in nominal voltage range of 100 to 240 VAC two 100 to 240 VAC 50 to 60 Hz F 3 15 A 250 V fast blo 5 x 20 mm IEC fuses Warning For continued protection against fire hazard replace fuses only with those of the same type and rating Unpacking and inspecting The detector is packed and shipped in one carton which contains the following items e Waters 2489 UV Visible detector startup kit which includes this guide Power cord e Release notes Unpacking To unpack the detector 1 Unpack the contents of the shipping carton As you unpack the carton check the contents to make sure you have received all items 2 Check the contents of the startup kit 3 Save the shipping carton for future transport or shipment Inspecting If you discover any damage or discrepancy when you inspect the contents of the cartons immediately contact the shipping agent and your local Waters representative Customers in the USA and Canada can report damage and discrepancies to Waters Technical Service 800 252 4752 Others can phone their local Waters subsidiary or Waters corporate headquarters in Milford Massachusetts USA or visit www waters com For complete information on reporting shipping damages and submitting claims
98. erface 3 25 Help screen Enter the desired wavelength for detection on channel A Range between 190 and 700 To exit the Help screen press Enter If online Help is not available for the function you are working on pressing gets no response Operating the detector Overview of detector operation Tip If you are operating the detector under the control of an external system you can program any parameters not controlled by the external data system at the front panel of the detector before the external system takes control Recommendation To prevent reabsorption of dissolved oxygen run the solvent degasser continuously when operating the detector at wavelengths less than 230 nm Requirement To maintain optimum system performance be sure to replace the front left hand panel cover before resuming normal operation of the detector Operating modes You can use the detector in either single or dual wavelength mode over a range of 190 to 700 nm The detector defaults to the mode of operation in effect when the instrument was last powered off Stand alone operation When using the detector as a stand alone instrument you can store as many as five methods containing as many as 50 timed and 2 threshold events each An asterisk in the method number field on the absorbance screen indicates current conditions not a stored method See page 3 34 for information on how to store a method 3 26 Preparing the Detector Remote cont
99. erium lamp does not light Faulty lamp Replace the lamp Lamp not plugged in Plug in the lamp connector Bad lamp power supply board Contact Waters Technical Service Lamp switch in the off position Inspect the rear panel connections or timed event within a method Ethernet problems Bad Ethernet cable Replace the Ethernet cable Troubleshooting 5 21 General system troubleshooting Continued Symptom Front panel display fails to illuminate Possible cause Broken electrical connection Corrective action Inspect electrical connections Fuse blown Inspect and if necessary replace fuse s Bad LCD or control board Contact Waters Technical Service Front panel displays odd characters Faulty EPROMs Bad LCD control board Contact Waters Technical Service In dual wavelength mode time scale is incorrect Data rate on controller is gt 1 pt sec In dual wavelength mode you must select a data sampling rate of 1 point per second Keypad not functioning Keypad defective 1 Cycle power to the detector and run the keypad diagnostic test 2 Contact Waters Technical Service No sample and reference energy Lamp burned out Attempt to reignite using the lamp key Replace the lamp Lamp extinguished Check the lamp icon Run the Sample amp ref energy diagnostic test Peak out of range
100. error at start up Cuvette in cuvette holder mobile phase absorbance too high air bubbles 1 Remove cuvette from cuvette holder 2 Be sure the flow cell mobile phase does not absorb above 250 nm 3 Ensure that no air bubbles are in the flow cell 4 Recalibrate the detector If problems persist contact Waters Technical Service 5 22 Error Messages Diagnostic Tests and Troubleshooting Safety Advisories Waters instruments display hazard symbols designed to alert you to the hidden dangers of operating and maintaining the instruments Their corresponding user guides also include the hazard symbols with accompanying text statements describing the hazards and telling you how to avoid them This appendix presents all the safety symbols and statements that apply to the entire line of Waters products Contents Topic Page Warning symbols A 2 Caution symbol A 5 Warnings that apply to all Waters instruments A 5 Electrical and handling symbols A 12 A 1 Warning symbols Warning symbols alert you to the risk of death injury or seriously adverse physiological reactions associated with an instrument s use or misuse Heed all warnings when you install repair and operate Waters instruments Waters assumes no liability for the failure of those who install repair or operate its instruments to comply with any safety precaution Task specific hazard warnings The following warning symbols alert y
101. error messages Continued Error Message Dark current too low 0 Description The dark energy level equals 0 Corrective Action 1 Cycle power to the detector 2 Contact Waters Technical Service Electronic A D failure Lamp optimization is adjusted at the minimum level Cycle power Data acquisition via A D converters is interrupt driven If interrupt is too long problem with data acquisition is indicated 1 Cycle power to the detector 2 Contact Waters Technical Service Filter initialization failure Erbium position Unit sensors cannot find erbium filter position 1 Cycle power to the detector 2 Contact Waters Technical Service Filter initialization failure No filters found Unit sensors observe transition to dark before homing the optical filter 1 Cycle power to the detector 2 Contact Waters Technical Service Filter initialization failure No reference energy Unit sensors cannot find any light energy before homing the optical filter 1 Cycle power to the detector 2 Contact Waters Technical Service Filter initialization failure No response Unit sensors cannot identify any dark regions 1 Cycle power to the detector 2 Contact Waters Technical Service Filter initialization failure Order filter position Unit sensors cannot find the order filter position 1 Cycle power to the detector 2 C
102. es changing 3 32 monitoring contact closures 5 15 lamp aging 3 28 mounting the detector 2 4 moving to the last entry in a list 3 12 N navigating in reverse order 3 10 negative number entry 3 12 new timed event 3 36 Next arrow 3 10 Next icon 3 6 Next key 3 10 noise filtering 1 7 3 16 numerical keys 3 10 O obtaining MaxPlot 3 34 RatioPlot 3 33 stored spectrum information 3 54 offset absorbance 3 13 Auto Zero Offset diagnostic test 5 11 voltage 3 13 operating as a stand alone instrument 3 26 detector 3 26 3 62 in dual wavelength mode 1 11 3 31 3 34 in single wavelength mode 1 10 1 11 3 30 3 31 modes 3 26 specifications B 1 B 5 trace and scale functions 3 20 3 22 under remote control 3 277 3 35 operator interface 3 4 optical and electronic design 1 4 component specifications B 3 detector specifications B 4 filter override diagnostic test 5 11 optics 1 4 optional flow cells 4 15 other equipment configuring with 1 2 connecting to 2 19 2 34 Ethernet connections 2 13 2 14 output connections 2 9 signals 2 12 P pace 3 42 parameters absorbance threshold timed event 3 36 analog out dual wavelength 3 19 AUFS 3 18 auto zero on inject 3 17 auto zero on inject 3 17 3 19 auto zero on wavelength changes 3 19 auto zero timed event 3 35 chart mark timed event 3 35 chart polarity 3 19 dual wavelength mode 1 12 filter time constant 3 19 lamp timed event 3 35 maximum ratio 3 19 minimum ratio 3 19
103. es necessary You should replace the lamp when the energy value approaches 15 nanoamps nA which corresponds to the cutoff adopted in the detector s diagnostic tests Ultimately however the detector s performance is a function Replacing the lamp 4 17 of its application so replace lamps whenever you determine doing so is appropriate for your method The detector s on board diagnostic tests allow the recording of lamp usage and reporting of the lamp serial number When to replace the lamp Rule Always run the Change Lamp diagnostic test after you install a new lamp see page 5 15 Replace the lamp when e it fails to ignite at startup e the energy level in the lamp causes a decrease in sensitivity to the point where the baseline 1s too noisy for your LC application Rule Always run the procedure in Recording sample and reference beam energies on page 3 28 when you change the detector lamp Waters warrants the lamp to ignite and pass startup diagnostic tests for 2000 hours or one year from the date of purchase whichever comes first Removing the lamp Warning The lamp housing gets extremely hot during operation To prevent burn injuries allow the lamp to cool for 30 minutes before removing it keep the lamp in the housing when handling the lamp Warning To avoid eye injury from ultraviolet radiation exposure power off the detector before changing the lamp wear eye protection that filters ultraviolet
104. etrahydrofuran 230 Ethyl sulfide 290 Toluene 285 Ethylene dichloride 230 Xylene 290 Mixed mobile phases The table below contains approximate wavelength cutoffs for some other solvents buffers detergents and mobile phases The solvent concentrations C 10 Solvent Considerations represented are those most commonly used If you want to use a different concentration you can determine approximate absorbance using Beer s Law because absorbance is proportional to concentration Wavelength cutoffs for different mobile phases 1 vial liter UV UV Mobile phase cutoff Mobile phase cutoff nm nm Acetic acid 1 230 Sodium chloride 1 M 207 Ammonium acetate 205 Sodium citrate 10 mM 225 10 mM Ammonium bicarbonate 190 Sodium dodecyl sulfate 190 10 mM BRIJ 35 0 1 190 Sodium formate 10 mM 200 CHAPS 0 1 215 Triethyl amine 1 235 Diammonium phosphate 205 Trifluoracetic acid 0 1 190 50 mM EDTA disodium 1 mM 190 TRIS HCl 20 mM pH 7 0 202 212 pH 8 0 HEPES 10 mM pH 7 6 225 Triton X 100 0 1 240 Hydrochloric acid 0 1 190 Waters PIC Reagent A 200 1 vial liter MES 10 mM pH 6 0 215 Waters PIC Reagent BG 225 1 vial liter Potassium phosphate Waters PIC Reagent B 6 190 monobasic 10 mM 190 low UV 1 vial liter dibasic 10 mM 190 Sodium acetate 10 mM 205 Waters PIC Reagent D 4 190 Wavelength selection for chromophore detection Cer
105. ette holder are located Site selection and power requirements Site selection Install the detector in an area that meets the requirements listed in the table below Installation site requirements Operating temperature 4 to 40 C 89 to 104 F range Site selection and power requirements 2 3 Installation site requirements Continued Parameter Requirement Storage temperature 30 to 60 C 22 to 140 F range Relative humidity 20 to lt 95 noncondensing Storage humidity range 0 to lt 95 noncondensing Bench space 12 7 cm 5 inches clearance at rear Vibration Negligible Static electricity Negligible Power Grounded AC 100 240 VAC 50 60 Hz Power cord type required SVT in the United States HAR type or better in Europe For information regarding the type of cord to use in other countries contact your local Waters distributor Requirement You must mount the detector on a level surface to allow proper function of the drip management system drain tube which you can connect to a waste reservoir to divert solvent leaks from the flow cell Power requirements Warning To avoid electric shock use the SVT type power cord in the United States and HAR type or better cord in Europe power off and unplug the detector before performing any maintenance procedures on the instrument connect all HPLC system components to a common ground The detector r
106. etter understand what you expect from our documentation so that we can continuously improve its accuracy and usability We seriously consider every customer comment we receive You can reach us at tech_comm waters com Contacting Waters Contact Waters with enhancement requests or technical questions regarding the use transportation removal or disposal of any Waters product You can reach us via the Internet telephone or conventional mail Waters contact information Contacting medium Information Internet The Waters Web site includes contact information for Waters locations worldwide Visit www waters com Telephone and fax From the USA or Canada phone 800 252 HPLC or fax 508 872 1990 For other locations worldwide phone and fax numbers appear in the Waters Web site Conventional mail Waters Corporation 34 Maple Street Milford MA 01757 USA Safety considerations Some reagents and samples used with Waters instruments and devices can pose chemical biological and radiological hazards You must know the potentially hazardous effects of all substances you work with Always follow Good Laboratory Practice and consult your organization s safety representative for guidance When you develop methods follow the Protocol for the Adoption of Analytical Methods in the Clinical Chemistry Laboratory American Journal of Medical Technology 44 1 pages 30 37 1978 This protocol addre
107. face 3 11 Detector keypad description Continued Key Unshifted Shifted Enters a decimal point H Some edit fields accept We Also positions the cursor at negative number entries Use the last entry in a list this function to invert the sign of the number in an active field CE Clears an editing Clear Field Clears the coe miara change and returns the current entry field before you CE contents of a field to its enter the desired values previous value Sets the value to a unique word for some fields For example in the voltage offset diagnostic display you can enter either a numeric offset value or press CE to change it to OFF Enter Completes the entry in an edit field Also advances the active field as if the Down arrow had been pressed except Eir after editing the wavelength on the absorbance screen Press Enter to acknowledge error messages and other prompts In these cases the word Enter appears as a cue in the lower right hand border of the message text Navigating the user interface To operate the detector 1 Press the Enter key or the up and down arrow keys to navigate among editable fields on a display Tip A thickened border highlights the active field Press Enter to advance the active field once you complete an entry When you make an error press CE Clear Entry to undo any changes and return to the active entry field Tip
108. ference beam energies on page 3 28 before pumping any mobile phase or solvent through the flow cell To pump solvent through the unit before initial use 1 Connect the detector to a data system or chart recorder see Chapter 2 for complete information on connecting the detector to external devices Using the operator interface 3 27 Power on the detector Result The front panel displays a series of initialization messages for about 5 minutes See page 3 2 When initialization ends the detector displays the absorbance screen Allow the detector to warm up for at least 30 minutes before operating Tip If the startup verification diagnostic tests fail note the error message to determine the corrective action and refer to Chapter 5 Recording sample and reference beam energies To determine baseline values on your detector for future reference and to monitor lamp aging decreased lamp output energy You must record the baseline sample and reference beam energies to compare against future readings Use these baseline values to troubleshoot the detector to determine whether the solvent is contaminated the flow cell is contaminated the lamp needs to be replaced there is an air bubble in flow cell To record the sample and reference beam energies 1 2 On the absorbance screen use the arrow keys to highlight the A field Enter 230 into the X field and then press Enter Ki 0 000182 Aso ln ae 2 0000 aurs
109. fines the minimum AU value for either wavelength A or B before the actual ratio is calculated Both absorbance values A and B must be above this value or 0 V is charted If both absorbances are above this value the absorbances are divided A B and charted The output voltage is scaled proportionally to the ratio depending on the selected channel minimum ratio and maximum ratio settings Minimum ratio An actual ratio equal to the minimum ratio value results in 0 V being charted Using the operator interface 3 15 Detector functions Continued Function Description Maximum ratio An actual ratio equal to the maximum ratio results in a full scale output of 2 V Absorbance offset is ignored with this selection For a RatioPlot the actual voltage charted is Volts out 0 Vif Absorbance A and B lt minimum AU Volts out Absorbance ratio minimum ratio X 2 V maximum ratio minimum ratio To ensure proper operation of the RatioPlot function be sure the selected time constants for both channels are set to the same value Difference Plot A B Charts the difference in absorbances at two different wavelengths The scaling for the difference plot is identical to the Absorbance selection above except that the charted absorbance is the difference subtracted in value of the two absorbances measured on the A and B wavelengths The detector uses the AUFS absorbance offset and voltage of
110. fset of the selected channel for scaling Volts out Absorbance difference A B x 2 VIAUFS of selected channel Filter time constant Adjusts the noise filter time constant to achieve the optimum signal to noise ratio without changing the sensitivity setting See the discussion Filtering noise on page 1 7 in for more information Voltage offset Adjusts the charted analog output signal Entered in millivolts this function adjusts the 2 V signal by the entered value This function is useful for making minor adjustments and for rendering null any offset between the detector and a connected external data system Chart polarity Inverts the charted chromatogram Entering the plus sign produces a normal chromatogram entering the minus sign produces an inverted chromatogram 3 16 Preparing the Detector Detector functions Continued Function Description Auto zero on inject Selected by default this parameter results in an auto zero each time the detector receives an inject start signal via contact closure Ethernet or through the front panel You can disable this parameter by pressing any numerical key Auto zero on X This parameter results in an auto zero each time a changes wavelength change is requested If you disable this function significant changes in measured absorbance can occur after each wavelength change Selecting to zero sets the signal level to zero Selec
111. g membrane is fixed in each vacuum chamber To increase the length of membrane you can connect two or more vacuum chambers in series The in line degasser is available as an option or factory installed in the Waters 2695 Separations Module XE model Wavelength selection This section includes UV cutoff ranges for common solvents common mixed mobile phases chromophores Wavelength selection C 9 UV cutoffs for common solvents The table below shows the UV cutoff the wavelength at which the absorbance of the solvent is equal to 1 AU for some common chromatographic solvents Operating at a wavelength near or below the cutoff increases baseline noise because of the absorbance of the solvent UV cutoff wavelengths for common chromatographic solvents Solvent aa Solvent ao 1 Nitropropane 380 Ethylene glycol 210 2 Butoxyethanol 220 Iso octane 215 Acetone 330 Isopropanol 205 Acetonitrile 190 Isopropyl chloride 225 Amyl alcohol 210 Isopropyl ether 220 Amyl chloride 225 Methanol 205 Benzene 280 Methyl acetate 260 Carbon disulfide 380 Methyl ethyl ketone 330 Carbon tetrachloride 265 Methyl isobutyl 334 ketone Chloroform 245 Methylene chloride 233 Cyclohexane 200 n Pentane 190 Cyclopentane 200 n Propanol 210 Diethyl amine 275 n Propyl chloride 225 Dioxane 215 Nitromethane 380 Ethanol 210 Petroleum ether 210 Ethyl acetate 256 Pyridine 330 Ethyl ether 220 T
112. he detector stand alone or remote control Types of instruments that make up your HPLC system This section describes the input output I O and digital signal connections that you can make from the two rear panel connectors and the Ethernet connector Installing the Detector The flow chart below provides an overview of the steps involved in making signal connections to the detector Overview of making signal connections Start signal connection procedure Connect to Install Ethernet and Ethernet bus inject start cables Connect to other Install event and devices UO cable s Signal connections complete Making electrical power connections 2 11 2 12 Making UO signal connections The rear panel includes two removable connectors that hold the pins for the I O signals as shown in the figure below These connectors are keyed so that you can insert them one way only UO signal inputs and outputs Connector 0 NNN UO signals Connector II Analog 1 Analog 1 Ground Analog 2 Analog 2 Switch 1 Switch 1 Ground Switch 2 Switch 2 URUVUVVOAH A DO DD JO Om SS Gab Inject Start Inject Start Ground Lamp On Lamp On Chart mark Chart mark Ground Auto Zero O Auto Zero The table below describes each of the signals available on the I O connectors See Appendix B for details on the electrical specifications for the signa
113. he flow cell within its assembly The detector is shipped with a standard analytical flow cell installed Replace the flow cell when the flow cell becomes damaged you want to use one of the optional flow cells see the table on page B 5 To prepare for replacing the flow cell 1 Unpack and inspect the new flow cell 2 Power off the detector and disconnect the power cord 3 Remove the front left hand panel cover Maintaining the flow cell 4 15 4 Disconnect the detector inlet outlet tubing from the main column connection and cap To replace the flow cell 1 OO SER EOS QD Using a 1 4 inch flat blade screwdriver loosen the three captive screws on the front plate of the flow cell assembly see the figure on page 4 9 Gently pull the assembly toward you Insert the new flow cell assembly into the detector Tighten the captive screws Reconnect the inlet outlet tubing to your LC system Reconnect the power cord and then power on the detector Replacing the lamp Recommendation Waters recommends that you change the detector lamp when it repeatedly fails to ignite or when the detector fails to calibrate See also The tables titled Start up calibration and operating error messages page 5 3 Instruments error messages page 5 6 and Detector diagnostic tests page 5 10 This section describes the procedure for removing and replacing the detector deuterium lamp Lamp characteristics Th
114. he operator interface 3 9 Detector keypad description Continued Key Unshifted Shifted Next Displays a screen with Previous When the Next key Previous additional options related to a is available use Previous to Next current screen Repeatedly navigate through the screens pressing this key brings back in the reverse order the screen on which you started On most screens where this key is active the NEXT arrow appears in the lower right hand corner of the display A B On screens with the A B METHOD Displays the list METHOD icon in the upper left hand of options for creating and A B corner this key toggles clearing timed and threshold between channel A and events storing retrieving channel B parameters and resetting methods DIAG Displays the choice CONFIGURE Displays the Wee list of diagnostic tests first Configuration screen TRACE Displays the Scale When the wavelength idi absorbance monitor trace for trace or spectrum screen is TRACE channel A or B visible this function permits modification of the display range in the X time or wavelength and Y absorbance dimensions Shift Enables the shifted functions identified by the text at Shift the top of most keys The shifted state is temporary and resets after the next keystroke 0 9 Enters a numeral from 0 9 See descriptions for
115. hermal changes 1 14 Theory and Principles of Operation Installing the Detector The detector requires connections to electrical power and to sample and waste lines to operate in any standard laboratory environment This chapter describes how to install the detector and connect it to the electrical supplies and to other equipment in an HPLC system Contents Topic Page Preparing for installation 2 2 Site selection and power requirements 2 3 Making plumbing connections 2 6 Making electrical power connections 2 9 Connecting to other devices 2 19 2 1 Preparing for installation Major steps in installing the detector Start installation procedure Make power connections Select appropriate site Make signal connections Unpack and inspect Make connections to other devices Install detector Installation complete Make plumbing connections After installing the detector verify its function and keep the verified chart output if applicable on file Verification ensures proper operation of the detector optics and electronics For proper verification procedures see Verifying that the detector operates properly on page 3 27 2 2 Installing the Detector Detector dimensions 20 8 cm 8 2 inches Me 50 3 cm 19 8 inches Caution Do not access the instrument through the top cover but through the front left hand panel where the lamp housing flow cell assembly and cuv
116. iagnostic test 1 From the Lamp display amp keypad choice list press 2 Test keypad to run the keypad test Keypad diagnostic test display L1 C Press Enter twice to exuit 2 Onthe keypad diagnostic test display press any key to begin the test Then press each key until you pressed all of them If the keypad 1s operating properly each key location 1s filled in and then cleared with another press of the key If any key does not respond when pressed contact your Waters service representative Rule You must press Enter twice to exit the keypad diagnostic display To run the test display diagnostic test 1 From the Lamp display amp keypad choice list press 3 Test display to run the test Result The display fills from top to bottom and from right to left then returns to the Lamp display amp keypad choice list If the display does not completely fill either horizontally or vertically contact your Waters service representative 2 From the Lamp display amp keypad choice list press 4 Using the other detector diagnostic tests The user diagnostic test display provides two additional tests Generate test peaks Specifies the generation of test peaks to calibrate your chart recorder or data system Manually override the optical filter Selects a filter different from that specified as part of the detector s normal operating mode User selected diagnostic tests 5 17 To run
117. ibration replaces the previous calibration data with new data See Wavelength calibration on page 3 30 for the manual wavelength calibration procedure The verification and calibration algorithms are virtually identical However the verification algorithm can issue an error message indicating that actual data do not match stored data where the calibration algorithm replaces the stored data with the new The detector wavelength verification procedures establish an approximate Home position using a grating homing sensor Once Home is established the detector locates and references the 656 1 nm peak in the deuterium lamp emission spectrum The integral erbium filter moves into the common light path ahead of the flow cell entrance slit enabling the detector to locate three additional spectral features at these wavelengths 256 7 nm UV 879 0 nm 521 5 nm The verification tests for the detector require five minutes of lamp warmup time If you run the detector continuously Waters recommends that you perform wavelength verification weekly by turning off the detector then turning it on again See Wavelength calibration on page 3 30 Principles of operation 1 9 Operational modes The detector operates in single or dual wavelength mode allows spectrum scanning using a flow cell or a cuvette and provides RatioPlot difference plot and MaxPlot functions Single wavelength mode Single wavelength is the default mod
118. ical mirror Filter wheel Shutter wavelength calibration filter and second order filter Entrance slit Blazed plane holographic diffraction grating Beamsplitter Sample and reference photodiodes Waters TaperSlit Flow Cell its entrance is the exit slit of the monochromator Cuvette holder 1 4 Theory and Principles of Operation Waters 2489 UV Visible detector optics assembly Spherical mirror Ellipsoidal mirror Optional Grating Sample photodiode Reference photodiode Optics assembly light path The detector provides an extremely efficient design for exceptionally high light throughput It operates as follows 1 The ellipsoidal mirror collects light from the lamp and focuses it through the filter wheel onto the entrance slit The spherical mirror directs light toward the grating A different portion of the spherical mirror focuses dispersed light of a particular wavelength band determined by the grating angle onto the entrance of the flow cell Light exiting the flow cell passes through the cuvette location to the sample photodiode 2 The beamsplitter located just ahead of the flow cell diverts a portion of the light to a reference photodiode 3 When you enter a new wavelength through the detector s front panel or through Empower or MassLynx software the detector rotates the grating to the appropriate position Principles of operation 1 6 4 The preamplifier board integra
119. ine maintenance To achieve optimal performance replace solvent reservoir filters in your HPLC system regularly filter and degas solvents to prolong column life reduce pressure fluctuations and decrease baseline noise flush buffered mobile phases out of the detector with HPLC grade water followed by a 5 to 10 methanol solution each time the detector is powered off This process prevents plugging of the solvent lines and flow cell damage to the components microbial growth Maintaining the flow cell A dirty flow cell can cause baseline noise decreased sample energy levels calibration failure and other problems with operation of the detector There are two phases in cleaning the flow cell Flushing Removing and cleaning If flushing is ineffective remove and clean the flow cell Replace flow cell components as required Caution Always replace the flow cell gaskets when cleaning rebuilding or replacing other flow cell components This section provides information about the following procedures Flushing the flow cell Removing and cleaning the flow cell Disassembling and reassembling the flow cell Maintaining the flow cell 4 5 Flushing the flow cell 4 6 Flush the flow cell when it becomes contaminated with the residues of previous runs and after each detector shutdown A dirty flow cell can cause baseline noise decreased energy levels calibration failure and other problems
120. ing proper use and handling Guidelines are provided in the latest edition of the National Research Council s publication Prudent Practices in the Laboratory Handling and Disposal of Chemicals Caution symbol The caution symbol signifies that an instrument s use or misuse can damage the instrument or compromise a sample s integrity The following symbol and its associated statement are typical of the kind that alert you to the risk of damaging the instrument or sample Caution To avoid damage do not use abrasives or solvents to clean the instrument s case Warnings that apply to all Waters instruments When operating this device follow standard quality control procedures and the equipment guidelines in this section Caution symbol A 5 A A 6 Attention Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment Important Toute modification sur cette unit n ayant pas t express ment approuv e par l autorit responsable de la conformit la r glementation peut annuler le droit de l utilisateur exploiter l quipement Achtung Jedwede Anderungen oder Modifikationen an dem Ger t ohne die ausdr ckliche Genehmigung der f r die ordnungsgem e Funktionst chtigkeit verantwortlichen Personen kann zum Entzug der Bedienungsbefugnis des Systems f hren Avvertenza qualsiasi modifica o alterazione ap
121. ion Contrast Previous Cancel and Clear Field Using the operator interface 3 7 3 8 Keypad SCAN AAA Reset HOME Chart mark Auto Zero Run Stop METHOD Lamp Lock Calibrate A B 1 2 3 CONFIGURE System Info Contrast DIAG 4 5 6 Scale TRACE 7 8 9 Cancel j Clear Field 0 D CE A Y Previous Next Primary function keys take effect immediately with no further entry required Secondary function keys require you to enter information into parameter fields and to press Enter for the functions to take effect Keys that appear in all caps HOME DIAG TRACE METHOD CONFIGURE and SCAN directly invoke a function from most screens For numerical entries from 1 to 9 on choice lists or menus enter the number corresponding to the desired item and then press Enter For the number 10 select 0 and then press Enter To go to the end of a choice list select e For entries numbered 11 or 12 scroll to the desired 1tem on the choice list and then press Enter Preparing the Detector The table below presents the functions of the primary and secondary keys Detector keypad description lower right corner of the absorbance screen Initiates scans Key Unshifted Shifted HOME Displays the Displays context sensitive absorbance screen containing help when available HOME the icons and the Wavelength and AUFS fields Chart mark Causes a SCAN
122. light keep the lamp in the housing during operation To remove the lamp Extinguish the lamp using the keypad by pressing Shift Lamp and then pressing Shift Lamp again Tip Using the keypad to extinguish the lamp allows the lamp to cool faster because of the operation of the instrument s cooling fan To 4 18 Maintaining the Detector power off the lamp using a timed event see the instructions in the Empower or MassLynx Help Power off the detector and disconnect the power cord After extinguishing the lamp allow it to cool for at least 30 minutes Remove the front left hand panel cover as described in Proper operating procedures on page 4 4 Disconnect power to the lamp and disconnect the cord Lamp assembly and power connector Power connector Replacing the lamp 4 19 6 Loosen the two captive screws in the lamp base Loosening the captive screws at the lamp housing base 7 Withdraw the lamp assembly from the lamp housing 4 20 Maintaining the Detector Caution Lamp gas is under slight negative pressure To prevent shattering the glass use care when disposing of the lamp Cushion the old lamp by placing it in the packaging material of the new lamp before disposal Removing the lamp Installing the new lamp Warning To avoid exposing your eyes to harmful ultraviolet radiation never ignite a lamp while it is outside the instrument or if it 1s not properly
123. llowing filter choice list Automatic Second Order None Erbium Shutter OU Coco N eR Tip The detector normally operates with the filter in the Automatic position This is a sticky diagnostic test Press the number corresponding to your filter choice or leave the default filter choice Automatic on To deactivate this diagnostic test press DIAG and then press 1 or select Automatic from the choice list Service diagnostic tests Only qualified Waters service personnel can access the detector service diagnostic tests User selected diagnostic tests 5 19 Troubleshooting This section provides some causes of errors and recommended troubleshooting actions Keep in mind that the source of apparent detector problems may lie within the chromatography or your other instruments as well as the detector Most detector problems are relatively easy to correct If you are unable to correct a problem or a failed condition contact Waters Technical Service When you contact Waters To expedite your request for service ensure the following information is available when you call Waters Technical Service Detector serial number Problem symptom s Operating wavelength s AUFS or measurement range Flow rate Filter setting Type of column Operating pressure Solvent s System configuration other components Tip The detector can be configured as part of a system
124. lly 1 Press Calibrate Shift 3 from the detector keypad Result A message appears asking whether you removed the cuvette and flushed the flow cell with a transparent solvent Waters recommends methanol or water 2 Press Enter to continue the calibration cycle or Cancel to invoke the absorbance screen without calibrating the detector Result After you press Enter the detector cycles through the calibration procedure and briefly displays a series of initialization messages similar to those you saw at startup see page 3 2 If calibration is successful the detector beeps three times and displays the maximum error in nanometers of the calibration shift farthest from the last calibration 3 Press Enter to complete calibration Result A Calibration complete message appears momentarily Other messages such as Optimizing system performance and Restoring last setup can appear before the display returns to the absorbance screen If calibration is successful the error message lt Error gt that was displayed on the absorbance screen before you recalibrated the detector disappears 4 If calibration is unsuccessful retry cycle power to the detector or see Chapter 5 Operating the detector in single wavelength mode The detector is optimized for single wavelength operation the default operating mode 3 30 Preparing the Detector To invoke single wavelength mode 1 From the absorbance or HOME sc
125. losure signal to command the detector to perform an auto zero at the start of an injection Auto zero connections To auto zero the detector at the start of an injection make the connections described in the table and illustrated in the figure below Use any available pair of Inject Start terminals on the autosampler Autosampler and detector auto zero connections 2489 detector A inputs 717plus autosampler terminal Pin 9 Auto Zero Inject Start any one of three paired with Pin 10 Auto Zero Inject Start any one of three paired with Autosampler auto zero connections to the detector 717plus connector 2489 connector II oe ez 1 Inject Start 92 2 Inject Start 12 Pom oe 3 Ground 11 Start Oz 4 Lamp On 10 Pom ole 5 Lamp On 9 Start Olo 6 Chart mark Inject 7 Chart mark 7 Start Q r 8 Ground 6 Inject O e 9 Auto Zero Start Qo 10 Auto Zero Ol Oloa Ola Le Inject start connections You can also use the Inject Start connections on the autosampler to connect to the Inject Start signals on the detector to program the start of the active method 2 32 Installing the Detector To program an inject start make the connections described in the table and illustrated in the figure below Use any available pair of Inject Start terminals on the autosampler Autosampler and detector inject start connections 2489 detector Il
126. ls UO signals for the detector Signal Inject Start Description conditions apply immediately TTL contact closure Configurable input to initiate sequencing of time programmed events Defines the start of a run typically an injection and resets and starts the run time clock to 0 00 minutes Initial Lamp On Off the deuterium lamp off and on Configurable input to allow an external device to turn Chart mark Configurable input to add a chart mark at 1096 of full scale to either or both analog output channels Installing the Detector UO signals for the detector Continued Signal Description Auto Zero Configurable input to auto zero either or both channels Analog 1 2 V full scale analog output signal of channel A scaled to the current AUFS setting Analog 2 2 V full scale analog output signal of channel B scaled to the current AUFS setting Switch 1 2 Used to connect to a fraction collector Can be Switch 2 2 controlled by threshold and timed events a Inject Start Chart mark Auto zero and Lamp inputs are configurable from the detector first Configuration screen by setting the appropriate parameter to High See Configuring event inputs contact closures on page 3 23 for more detail b See the discussion of attenuation in the detector analog outputs in Making electrical power connections on page 2 9 Making Ethernet connections The
127. m and the current method number or an asterisk indicate current conditions are not stored as a method Bog Bre Ethernet control Indicates that the detector is controlled by a data system Numerical Run time Displays the time elapsed since you field minutes pressed Run or since an inject start signal was received TG Next Indicates that pressing Next displays additional screens Preparing the Detector Absorbance and message screen icons Continued Icon or field Icon field name Function Message screen Indicates an error message D icon Message screen Indicates a question icon Message screen Indicates a warning message icon Message screen Indicates information is being displayed Ck icon GS Message screen Indicates that you must standby icon Using the keypad The detector keypad shown below consists of 24 keys providing full numeric entry 10 digits plus a decimal point Enter Shift CE Clear Entry Next and Help functions s and t used for navigation only pressing s can also move the highlighted area to the left t to the right A B for channel selection navigation to specific screens HOME or absorbance DIAGnostics TRACE CONFIGURE METHOD and SCAN primary function keys Chart mark Auto Zero and Run Stop secondary function keys Scale Single or Dual wavelength Reset Clock Lamp Lock Calibrate System Informat
128. m Refer to the figure on page 3 43 which shows two scans of anthracene overlaid one at 100 nm min and one at 1000 nm min The higher the value you enter in the Pace field the lower the resolution of the scan Scanning spectra 3 47 6 Press Next 7 On the third zero scan parameter screen enter the AUFS value and then press Enter Tips To specify tick marks enter a number from 10 nm to 100 nm and then press Enter Toclear tick marks press CE See page 3 44 for examples of scans made with and without tick marks 8 Press Run to start the zero scan or press Next to return to the first zero scan parameter screen to verify parameter values and then press Run After the detector runs the zero scan it returns to the Scan choice list 3 48 Preparing the Detector Zero and sample scan screen Press to start scan 71 Tape Zero scan z A 254 380 nm 2 000 AUFS Face 100 nm min lord Mark each 10 nm Press to start scan 7 Type Sample scan J X 254 380 nm 2 000 AUFS Face 100 nm min 1or2 Mark each 10 nm HENT Zero scan screen 1 of 4 Kaes Ee to Pace Zotd 100 Inm min m Sample scan screen 1 of 3 AUFS 2 00000 Mark nl Ofm zorz egt Sample scan Screen 2 of 3 aurs 200000 Mark each 1 inm Zei PIT Press GIA to start scanning now Za foal m l Zero scan screen 3 of 4
129. mi prep cell 2 6 3 0 040 0 040 1000 70 Microbore cell 2 6 3 0 005 0 005 1000 70 Inert titanium cell 10 10 0 010 0 010 1000 70 High pressure cell for 10 10 0 009 0 009 3000 210 LCMS Variable path length 0 69to 0 15 3 0 04 0 04 1000 70 flow cell VPF 13 72 factory preset at 0 5 mm Autopurification cell 2 6 1 0 0 009 IN1 2000 140 0 020 IN2 0 040 OUT Optional Waters TaperSlit flow cell specifications B 5 B 6 Specifications Solvent Considerations Contents Topic Page Introduction C 2 Solvent miscibility C 3 Buffered solvents C 6 Head height C 6 Solvent viscosity C 6 Mobile phase solvent degassing C 7 Solvent degassing methods C 8 Wavelength selection C 9 C 1 Warning To avoid chemical hazards always observe Good Laboratory Practices when operating your system Introduction Preventing contamination For information on preventing contamination refer to Controlling Contamination in Ultra Performance LC MS and HPLC MS Systems part number 715001307 or visit www waters com Clean solvents Clean solvents provide reproducible results and permit you to operate with minimal instrument maintenance A dirty solvent can cause baseline noise and drift It can also block the solvent filters with particulate matter Solvent quality Use HPLC grade solvents for the best possible results Filter solvents through 0 45 um filters before their use Solvents distilled in glass generally mai
130. mperature application consider the effect of the higher temperature on solvent solubility buffers dissolved in water can precipitate when mixed with organic solvents Solvent miscibility C 3 When you switch from a strong buffer to an organic solvent flush the buffer out of the system with distilled water before you add the organic solvent Solvent miscibility on Boilin Miscibilit d Solvent Mec point c number d bat i 1 atm M 0 3 N decane 0 92 174 1 29 0 4 Iso octane 0 50 99 2 29 210 0 0 N hexane 0 313 68 7 29 0 0 Cyclohexane 0 98 80 7 28 210 1 7 Butyl ether 0 70 142 2 26 1 8 Triethylamine 0 38 89 5 26 2 2 Isopropyl ether 0 33 68 3 220 2 3 Toluene 0 59 100 6 23 285 2 4 P xylene 0 70 138 0 24 290 3 0 Benzene 0 65 80 1 21 280 3 3 Benzyl ether 5 33 288 3 3 4 Methylene chloride 0 44 39 8 20 245 3 7 Ethylene chloride 0 79 83 5 20 3 9 Butyl alcohol 3 00 117 7 3 9 Butanol 3 01 177 7 15 4 2 Tetrahydrofuran 0 55 66 0 17 220 4 3 Ethyl acetate 0 47 77 1 19 260 4 3 1 propanol 2 30 97 2 15 210 4 3 2 propanol 2 35 117 7 15 4 4 Methyl acetate 0 45 56 3 15 17 260 4 5 Methyl ethyl ketone 0 43 80 0 17 330 4 5 Cyclohexanone 2 24 155 7 28 210 4 5 Nitrobenzene 2 03 210 8 14 20 4 6 Benzonitrile 1 22 191 1 15 19 4 8 Dioxane 1 54 101 3 17 220 CA Solvent Considerations
131. mple and reference energies appear If you are operating the detector in dual wavelength mode press the A B key to view sample and reference energy on the other wavelength Using the input and output diagnostic tests You use the input and output diagnostic tests to display and reset the autozero offsets fix set absorbance fix set the voltage on the 2 V output monitor contact closures and toggle event switches generate test peaks override the optical filter 5 12 Error Messages Diagnostic Tests and Troubleshooting To perform any of the input and output diagnostic tests press DIAG and then press 3 for Input amp output A choice list appears Input amp output diagnostic test choice list Auto zero offsets z Fix absorbance a Fis voltage 4 Contact closures amp events Previous choices lt lt Displaying auto zero offsets From the Input amp Output choice list press 1 Auto zero offsets This diagnostic test also allows you to display and reset the offset to zero on both channels by pressing Cancel Shift 0 Auto zero offsets diagnostic display Channel A 0 2131 AU Channel B 12131 AU Press Sg to reset to zero offset on both channels Auto Zero Offsets ER Setting fixed absorbance value From the Input amp Output choice list press 2 Fix absorbance to set a fixed absorbance value for channel A or channel B The allowable range is from 4 0000 AU to
132. ms using Ethernet 2 13 2 14 Waters 600 series pump 2 28 2 31 Waters 717plus Autosampler 2 31 2 33 Waters 745 745B 746 Data Module 2 23 2 25 conserving lamp life 3 60 contact closures and events diagnostic test 5 11 configuring event inputs 3 23 monitoring 5 15 contacting Waters Technical Service 2 5 4 2 5 20 contamination flow cell 3 28 solvent 3 28 contamination preventing C 2 context sensitive help 3 25 contrast adjusting 3 24 changing 3 11 function 3 24 Contrast key 3 11 controlling from Waters data systems 2 13 cover removing 4 4 current method conditions 3 13 3 34 3 41 cuvette description 3 56 removing 3 59 replacing holder 3 59 cuvette holder illustrated 3 57 removing 3 58 replacing 3 59 cuvette option description 1 13 qualification features 1 3 sample analysis 1 3 scanning 3 42 3 56 3 59 using 1 4 D damage to the detector 2 5 to the flow cell 4 15 damage reporting 4 2 data system control 2 13 decimal point key 3 12 default Ethernet address 3 22 degassing benefits C 8 considerations C 9 solvents 3 26 4 5 deleting a timed event 3 37 design electronic 1 4 optical 1 4 detector as spectrophotometer 1 3 damage 2 5 Index 3 description 1 2 1 3 diagnostic tests 5 9 5 19 mounting 2 4 operating 3 26 3 62 operating under remote control 3 35 optics 1 5 powering off 3 62 setting up to run 3 13 site requirements for 2 3 specifications B 1 B 5 startup procedures 3 2 troubleshooting 5
133. n 6 Reconnect the power cord to the power entry module 4 26 Maintaining the Detector Error Messages Diagnostic Tests and Troubleshooting The detector provides both user and service diagnostic tests to troubleshoot system problems Contents Topic Page Error messages 5 2 User selected diagnostic tests 5 9 Troubleshooting 5 20 Error messages Startup error messages Startup diagnostic tests run automatically when you power on the detector They verify the proper operation of the detector electronics If one or more of the tests fail the detector beeps and displays an error message For serious errors it displays the word Error in brackets Error in place of the run time absorbance on the absorbance screen Tip To reduce the likelihood of errors be sure the cuvette holder is empty the flow cell contains degassed transparent solvent methanol or water and the front left hand panel cover is attached securely The tables in this section are organized as follows Messages requiring you to perform corrective action including messages encountered at startup and during calibration or operation e Messages requiring you to cycle power and then contact Waters Technical Service personnel if an error persists see Contacting Waters technical service on page 4 2 Most of these errors arise on startup 5 2 Error Messages Diagnostic Tests and Troubleshooting The table below provides startup c
134. n Installing the Detector To make inject start connections between the pump and the detector to start a method 1 Using a signal cable make the connections between the detector and the pump that are summarized in the table and illustrated in the figure below 2 Program the pump to provide a pulse output on the selected switch at the beginning of each run See also Waters 600E Multisolvent Delivery System User s Guide Pump and detector inject start connections 2489 detector Il Pin 1 Inject Start 600 series pump terminal S1 S2 or S4 Pin 2 Inject Start GND one of four a You can also connect the pump inject pin to the Pin 1 Inject Start on the detector and the inject ground pin to the Inject Start on the detec tor Pump inject start connections 600 series pump CHART PRESSURE SWITCHES 2489 connector Il ei KA 3 Red CHART PRESSURE 1 2 3 A 5 6 7 8 9 1 Inject Start Inject Start Ground Lamp On Lamp On Chart mark Chart mark Ground Auto Zero O Auto Zero Connecting the detector to the Waters 717plus autosampler The Waters 717plus autosampler signals the start of an injection through a contact closure signal on its Inject Start terminals You can use this contact Connecting to other devices 2 31 c
135. nal cable make the connections between the pump controller and the detector that are summarized in the table and shown in the figure below Pump and detector lamp on off connections 2489 detector Il 600 series pump terminal Pin 4 Lamp On Off S1 S2 or S4 Pin 5 Lamp On Off GND Pump lamp on off connections 600 series pump 2489 connector II CHART PRESSURE a a o I 1 Inject Start 2 Inject Start 3 Ground d Lamp On 5 Lamp On 6 Chart mark 7 8 9 1 CHART l PRESSURE Chart mark Ground Auto Zero O Auto Zero Installing the Detector Auto zero connections To make auto zero connections between the detector and the pump 1 Using a signal cable make the connections between the detector and the pump that are summarized in the table and illustrated in the figure below 2 Program the pump to provide a pulse output on the applicable switch S1 S2 or 84 at the beginning of each run See also Waters 600E Multisolvent Delivery System User s Guide Pump and detector auto zero connections 2489 detector Il 600 series pump terminal Pin 9 Auto Zero S1 S2 or S4 Pin 10 Auto Zero GND one of four Pump auto zero connections 600 series pump 2489 connector Il CHART 9 SWITCHES 4 PRESSURE Q le x 8 Red 1 Inj
136. nd the cuvette the solvent conditions in the flow cell must be identical for both scans See Scanning using the cuvette on page 3 56 Operational modes 1 13 RatioPlot The detector allows ratio plotting comparing the absorbances of a compound or analyte at two wavelengths The RatioPlot divides absorbances at two selected wavelengths and plots the resulting ratio on a chart recorder or data system over one output channel channel A Use the RatioPlot when detecting hidden components within individual peaks The RatioPlot of a spectrally homogeneous peak appears as a rectangular wave The RatioPlot of an impure peak appears as a distorted wave When obtaining a RatioPlot you must be operating the detector in dual wavelength mode the RatioPlot is output on the selected channel See page 3 33 MaxPlot The MaxPlot function monitors absorbance at two selected wavelengths and plots the maximum absorbance value for each sample component To obtain a MaxPlot you must operate the detector in dual wavelength mode The MaxPlot outputs the greater of the two absorbance values on the selected channel For an explanation of the MaxPlot procedure see the discussion Obtaining a MaxPlot on page 3 34 Thermal wander management To mitigate thermal instability caused by ambient temperature changes the the detector s variable speed fan runs at higher or lower speeds as needed The fan normally changes speeds in response to the t
137. nditions so that the original recalled method is no longer in effect causing the method number to change to an asterisk The operating parameters at the time of system shutdown are restored however any timed events or thresholds associated with the method are deactivated when power is restored On startup you always see an asterisk inside the method icon on the absorbance screen 3 34 Preparing the Detector When the detector is operating under remote control that is control by Empower or MassLynx software the remote icon incorporating the letter E appears see the table on page 3 5 Timed events You can program as many as 50 timed events to the nearest 0 01 minutes As you enter timed events each new event appends to the end of the timed event list If you enter a time that is not in sequence with the events entered previously the timed event list sorts automatically when you press Next The detector allows programming of the timed events shown in the table below Caution Changing the sensitivity AUFS setting affects the 2 V output For example 1 AU gives 0 5 AU V and 2 AU gives 1 AU V Timed event parameters 4 Rect wave Number Event Units Range or default SEIN channel 1 Wavelength nm 190 to 700 Yes 2 Filter Seconds 0 Disable filter Yes time constant 0 0125 to 5 00 AA 0 5 to 5 0 3 Sensitivity AUFS 0 0001 to 4 0000 Yes 4 Chart mark Does not Does not apply Yes 10
138. nge The figure below shows the four highest peaks for the erbium scan shown in the graphical display above Sample erbium scan four highest peaks Range 1320 700 nm nm AU 1255 02228 2z 379 0 1931 3 523 01460 d B52 D1245 Press Next On the graphical display press Scale Shift TRACE to change the scale zoom in on one part of the display Tip You can alter the following four scaling parameters A1 minimum wavelength displayed 4 2 maximum wavelength displayed AUI minimum absorbance displayed The default is Auto AU2 maximum absorbance displayed The default is Auto Using this function you zoom in on various sections artifacts of the spectrum The scale of the spectrum is affected by the AUFS setting Press Next to advance through the four scaling parameters The figure below shows the sample scanned in the figure on page 3 50 The sample has been scaled by changing the wavelength parameters to 225 and 600 nm Scanning spectra 3 51 Sample erbium scan with I1 changed to 225 nm and D changed to 600 nm ZEE 27 9 Once you change one or more scaling parameters press Enter to reformat the graphical display 10 Once the scan reappears press Next to display the four highest peaks of the scaled scan Four highest peaks from the scaled sample erbium scan A Ronge 220 600 nm nm AU 12755 2228 2 379 0 1931 3 ban 01460 4241 0 0949
139. ntain their purity from lot to lot use them to ensure the best possible results Preparation checklist The following solvent preparation guidelines help to ensure stable baselines and good resolution Filter solvents with a 0 45 um filter Degas and or sparge the solvent Stir the solvent Keep in a place free from drafts and shock C 2 Solvent Considerations Water Use water only from a high quality water purification system If the water system does not deliver filtered water filter it through a 0 45 um membrane filter before use Using buffers When you use buffers dissolve salts first adjust the pH then filter to remove insoluble material Tetrahydrofuran When using unstabilized tetrahydrofuran ensure that your solvent is fresh Previously opened bottles of tetrahydrofuran contain peroxide contaminants which cause baseline drift Warning Tetrahydrofuran contaminants peroxides are potentially explosive if concentrated or taken to dryness Solvent miscibility Before you change solvents refer to the table below to determine the miscibility of the solvents to be used When you change solvents be aware that changes involving two miscible solvents may be made directly Changes involving two solvents that are not totally miscible for example from chloroform to water require an intermediate solvent such as isopropanol temperature affects solvent miscibility If you are running a high te
140. nur mit Sicherungen ersetzen deren Typ und Nennwert auf den Tafeln neben den Sicherungsabdeckungen des Ger ts gedruckt sind Attenzione per garantire protezione contro gli incendi sostituire i fusibili con altri dello stesso tipo aventi le caratteristiche indicate sui pannelli adiacenti alla copertura fusibili dello strumento Advertencia Para evitar incendios sustituir los fusibles por aquellos A del tipo y caracteristicas impresos en los paneles adyacentes a las cubiertas de los fusibles del instrumento EE Ay UK AL ERAR aif 1 PUB d Cas fai 25 TA ET ED ll ZA FERRI BEIENEE s YTRAKK NERS NAER S TT E ERI AREI TRES 22 A 2 SMO ASS SOAR 212 FA HHA 2E2E WSO Wa Av Ssvet ES 2 AA AEL FAS nHL 5 immo E a AHA CHEER E A AM ODIO MSIUVCWS 747133 05Ef0U aR E TIERS RSV Warnings that apply to all Waters instruments A 11 Electrical and handling symbols Electrical symbols These can appear in instrument user manuals and on the instrument s front or rear panels Electrical power on Electrical power off Standby C O Direct current Alternating current Protective conductor terminal Frame or chassis terminal Fuse Recycle symbol Do not dispose in municipal waste bai 3 D A 12 Safety Advisories Handling symbols These handling symbols and their associated text can a
141. nute scaled trace similar to that shown in the preceding figure with the starting absorbance or AU1 changed from auto to 1 T1 remains at 60 T2 remains at 0 Scaled trace for 60 minutes of continuous injections with AU1 changed to 1 A E rJ 1 min 1 40 Using the operator interface 3 21 The figure below shows a 60 minute trace on channel B scaled to the last 45 minutes T1 is changed to 45 Scaled trace changing T1 to 45 min 40 20 o As you modify the output using the Scale function the Trace function continues to display the detector output in real time on either or both channels Operating other detector functions Configuring the detector To change default configurations use the Configuration screens Press the CONFIGURE key Shift DIAG The first of three Configuration screens appears Tip Other functions such as specifying event inputs and enabling pulse periods are activated from the Configuration screens Configuration screens 7 disable shutter idle made Inject Chart mark Switohed Output Period Low E sinale rD Jec Auto zero Lamp 1or3 Fort Rectangular 3or3 HENT Low B Ignore E mg w ve n2 Leer HERT Configuration screen 1 of 3 Configuration screen 2 of 3 Configuration screen 3 of 3 Requirement To prevent acquisition of incorrect data when operating the detector in dual wavelength mode under the control of
142. o input to auto zero absorbance readings on channel A and or channel B You determine the response of the channel using the enable auto zero function explained in the table on page page 3 18 and shown in the figure on page 3 18 High Auto zero the channel when contact closure changes from off open to on closed Low Auto zero the channel when contact closure changes from on closed to off open Ignore Do not respond to auto zero input Using the operator interface 3 23 3 24 Lamp You can configure the lamp input level to turn the deuterium lamp on or off from an external device as follows High Turn lamp off when contact closure is on closed Low Turn lamp off when contact closure is off open Ignore Do not respond to Lamp input Setting pulse periods You use the third Configuration screen see page 3 22 to set pulse or signal width or to activate a pulse or rectangular wave on SW1 or SW2 Single pulse in seconds If SW1 or SW2 is programmed to generate a pulse as a timed or threshold event then the period of the signal single pulse width is as specified in this field range is 0 1 to 60 seconds Rectangular wave in seconds If SW1 or SW2 is programmed to initiate a rectangular wave as a timed or threshold event then the period of the signal the width of one pulse period in a rectangular wave or pulse train is as specified in this field range is 0
143. offset parameter 3 13 RatioPlot function 3 15 threshold events 3 36 3 39 threshold timed event parameter 3 36 trace 3 10 absorbance screen display 3 3 3 4 3 9 error message 5 5 icons 3 5 navigating from 3 13 secondary functions 3 14 accessing secondary functions 3 13 activating a pulse or rectangular wave 3 24 active method 3 40 additional functions in dual wavelength mode 3 31 additional peaks 3 32 adjusting analog signal 3 16 contrast 3 24 advancing to the next field 3 12 algorithms 1 9 analog outputs channel outputs 2 9 connections 2 20 dual wavelength 3 15 dual wavelength signal 3 19 signal adjusting 3 14 single wavelength 3 14 Specifications 3 13 analytes additional information on 1 11 comparing 1 14 dissolved 1 13 in cuvette operations 1 13 Anthracene 3 43 3 53 audience and purpose v AUFS parameter 3 18 sensitivity 3 44 auto zero connections to 600 series pump 2 29 function 3 17 on inject parameter 3 17 Auto Zero key 3 9 Index 1 automatic second order filter 1 3 1 4 1 10 auto zero configuring 3 23 connections to 717plus autosampler 2 32 function 3 9 3 17 offset diagnostic test 5 11 5 13 on inject parameter 3 17 3 19 on wavelength changes 3 17 on wavelength changes parameter 3 19 timed event parameter 3 35 B bandwidth specification B 2 beamsplitter 1 4 benefits of degassing C 8 biohazard warning A 4 buffered mobile phase flushing 4 5 removing 3 62 buffered solvents C 6 burst warning
144. on Guide The following table summarizes the detector connections to the e SAT IN module Detector connections to a e SAT IN module 2489 detector I E Pin 1 Analog 1 white Pin 3 Ground black Channel 1 or 2 Pin 4 Analog 2 white Pin 8 Ground black Channel 1 or 2 Connecting the detector to a 745 745B 746 data module You can connect the detector to a Waters 745 745B 746 data module using the analog output connector on the rear panel of the detector The analog Connecting to other devices 2 23 connector provides 2 V output that is scaled to the AUFS sensitivity setting and the voltage offset setting Caution To prevent oversaturation of the signal from the detector to the integrator do not exceed the input voltage rating of the integrator To send the analog output signal from the detector to the data module use the cable provided in the 2489 Detector Startup Kit to make the connections summarized in the table below and illustrated in the figure below Detector connections to a data module 2489 detector I ee Pin 1 Analog 1 red Pin 3 Ground black B Pin 4 Analog 2 red i Pin 8 Ground black To minimize the chance of creating a ground loop that can adversely affect measurement connect the shield of the cable to the chassis ground at one end only 2 24 Installing the Detector Data module connections to detector channels A and B 2489 connector
145. onnector to the chart or A D device in a data collection system Once you retrieve a spectrum for playback the detector displays it graphically and you can adjust the AUFS Sample energy is not charted during a replay Tip If you adjust the AUFS on a spectrum for replay the adjusted spectrum displays only on the charted output not on the detector graphical display To replay a spectrum 1 Press SCAN Shift Chart mark 2 Press 6 Real time replay 3 Enter the storage slot number 1 to 3 of the spectrum you want to play back Tip The default is the last spectrum acquired 4 Press Enter Result After a pause to retrieve the selected spectrum the detector begins to play back the spectrum on the analog connection Then the spectrum appears graphically Scanning using the cuvette Using the cuvette option facilitates sample handling and verifying and qualifying the instrument The detector uses a standard 10 mm path length spectrophotometric cell quartz cuvette You insert the cuvette with one of the two frosted sides facing up in the cuvette holder and place it in the detector flow cell assembly 3 56 Preparing the Detector Detector cuvette holder with the cuvette inserted D Frosted sides of cuvette facing up and down Restriction Because the scan is actually a composite of both the contents of the cuvette and the flow cell you need to perform cuvette scans under identical flow cell conditions If
146. ontact Waters Technical Service Error messages 5 7 Instrument error messages Continued Error Message Filter initialization failure Shutter position Description Unit sensors cannot find the shutter position Corrective Action 1 Cycle power to the detector 2 Contact Waters Technical Service Grating initialization failure Backlash too high Backlash is the difference between the forward and reverse peak positions of deuterium at 656 nm If this difference is greater than 1 step the unit displays this message 1 Cycle power to the detector 2 Contact Waters Technical Service Grating initialization failure No home sensor Search for the home sensor failed 1 Cycle power to the detector 2 Contact Waters Technical Service Lamp data not found Stored lamp data are invalid Cycle power to the detector This action removes the error Method not found Stored method data are invalid Cycle power to the detector This action removes the error Scan not found Stored scan data are invalid Cycle power to the detector This action removes the error System cannot respond Error occurs while unit is positioning next wavelength or changing modes Occurs during initialization or calibration 1 Cycle power to the detector 2 Contact Waters Technical Service Error Messages Diagnostic Tests and Troubleshooting
147. ou to risks that can arise when you operate or maintain an instrument or instrument component Such risks include burn injuries electric shocks ultraviolet radiation exposures and others When the following symbols appear in a manual s narratives or procedures their accompanying text identifies the specific risk and explains how to avoid it Warning General risk of danger When this symbol appears on an instrument consult the instrument s user documentation for important safety related information before you use the instrument A Warning Risk of burn injury from contacting hot surfaces A Warning Risk of electric shock A Warning Risk of fire A Warning Risk of needle puncture A Warning Risk of injury caused by moving machinery A Warning Risk of exposure to ultraviolet radiation A Warning Risk of contacting corrosive substances A Warning Risk of exposure to a toxic substance A Warning Risk of personal exposure to laser radiation A 2 Safety Advisories Warning Risk of exposure to biological agents that can pose a serious health threat Specific warnings The following warnings can appear in the user manuals of particular instruments and on labels affixed to them or their component parts Burst warning This warning applies to Waters instruments fitted with nonmetallic tubing Warning Pressurized nonmetallic or polymer tubing can burst Observe these precautions when working a
148. our data system by attaching the cable to the LAC E interface Note The Ethernet cable is supplied with the Waters data system 2 Connect the other end of the cable to the Ethernet connector on the rear panel of the detector Caution The maximum total cable length between Ethernet devices in a system is 20 meters 65 feet The maximum recommended cable length between two Ethernet devices is 4 meters 13 feet Longer total cable lengths can cause intermittent Ethernet communication failures Ethernet connections to the detector in a Waters Empower system busLAC E or LAC E s ER Ethernet cable Ethernet connector Empower workstation ue 2489 detector Tip When connecting the detector to a data system all detector parameters not configurable by the data system in use defer to local control 2 14 Installing the Detector Starting a method To start a method on the detector at the start of an injection from the separations module make the connections summarized in the table below and illustrated in the figure below that 2695 separations module B inputs and outputs Pin 1 Inject Start 2489 detector Il Pin 1 Inject Start Pin 2 Inject Start Pin 2 Inject Start 2695 separations module connections to the detector for starting a method 2695 connector B Inject Start Inject Start Ground Stop Flow Stop Flow Hold Inject 1 Hol
149. portata a questa unit e non espressamente autorizzata dai responsabili per la conformit fa decadere il diritto all utilizzo dell apparecchiatura da parte dell utente Atencion cualquier cambio o modificaci n efectuado en esta unidad que no haya sido expresamente aprobado por la parte responsable del cumplimiento puede anular la autorizaci n del usuario para utilizar el equipo ES REA ER IE a RB T POR TN A ct fi HE AT A vi SE EAE vc RT RE Er i CREER E AE SHEA YER AAA IE ERIT HM rr WT AS XETT AAR SPURS FY ES E DET E EAR VEZ BE IEE ol vf ALUS AL d HE E Be fir EE na JZ ce ny 0 0p 0x IO o ge lo 10 02 lz go e zo 02 Ol gt C nit 1 as TER RABID o DAME ZR AGB SEU PICA OR CUETO b ANXIE 2 F amp LC ORR POC 72S nT etk23do ER Safety Advisories Warning Use caution when working with any polymer tubing under pressure A Always wear eye protection when near pressurized polymer tubing Extinguish all nearby flames Do not use tubing that has been severely stressed or kinked Do not use nonmetallic tubing with tetrahydrofuran THF or concentrated nitric or sulfuric acids Be aware that methylene chloride and dimethyl sulfoxide cause nonmetallic tubing to swell which greatly reduces the rupture p
150. ppear on labels affixed to the outer packaging of Waters instrument and component shipments tt Keep upright Keep dry Fragile bs E Use no hooks Electrical and handling symbols A 13 A 14 Safety Advisories Specifications This appendix lists individual operating specifications for the Waters 2489 UV Visible Detector as follows Operational specifications Optical specifications Specifications for the optional flow cells Contents Topic Page Operational specifications B 2 Optical specifications B 4 Optional Waters TaperSlit flow cell specifications B 5 B 1 Operational specifications Operational specifications Condition Wavelength range Specification 190 to 700 nm Bandwidth lt 5 nm Wavelength accuracy 1 0 nm Wavelength 0 1 nm repeatability Single dry noise lt 5 AU at 230 nm 1 second digital filter 30 second segments 10 Hz data rate analytical cell Wet noise lt 8 AU at 230 nm 1 second digital filter 30 second segments 10 Hz data rate 1 mL min flow of acetonitrile analytical cell Dual channel noise dry lt 35 AU measured at 230 nm running at 230 to 280 nm with 2 second filter at 1 Hz data rate dry analytical cell Dual channel noise lt 45 AU wet measured at 230 nm running at 230 to 280 nm with 2 second filter at 1 Hz data rate 1mL min flow of acetonitrile an
151. recording new lamp serial number 4 23 4 24 sample and reference energy 3 28 rectangular wave signal 3 24 reduction in resolution 3 43 reference energy 3 45 photodiode 1 4 remote control 3 277 3 35 removing buffered mobile phase 3 62 cuvette 3 59 cuvette holder 3 58 flow cell 4 7 flow cell assembly 4 9 front left panel cover 4 4 lamp 4 18 4 21 repeatability specifications B 2 replacing cuvette holder 3 59 flow cell 4 15 4 16 flow cell parts 4 5 4 13 front left panel cover 4 4 fuses 4 25 4 26 lamp 4 16 4 23 solvent reservoir filters 4 5 replaying a spectrum 3 56 reservoirs positioning C 6 reset diagnostic tests 5 10 reset instrument 5 10 Reset key 3 9 resetting run clock 3 9 stored method 3 41 resolution reduction in 3 43 retrieving a method 3 40 returning to initial conditions 3 9 reviewing a scan 3 55 routine maintenance 4 5 run clock stopping 3 9 run time icon 3 6 Run Stop key 3 9 running new scan 3 46 3 53 sample scan 3 50 solvent degasser 3 26 zero scan 3 477 S safety advisories A 1 safety considerations maintenance 4 3 sample and reference energy diagnostic test 5 10 5 11 sample energy 3 28 3 45 sample photodiode 1 4 sample scan definition 1 13 parameters 3 47 procedure 3 45 3 49 running 3 50 screens 3 49 when to run 3 45 scale function 3 20 3 22 zooming 3 51 Scale key 3 10 3 20 scaling factor 3 10 SCAN key 3 9 3 45 Scanning subtracting 3 55 scanning
152. reen press the A AA key Shift Auto Zero if the detector 1s in dual wavelength the wavelength icon shows AA mode Result The detector displays a message indicating it 1s switching to single wavelength operation Enter the wavelength and sensitivity on the absorbance screen as well as any secondary parameters and timed or threshold events Caution Changing the sensitivity AUFS setting affects the 2 V output Select a second sensitivity setting while in single wavelength mode by pressing A B and specifying the appropriate AUFS values on the channel B screen Result A single wavelength is tracked on channel A so that you can use channel B to monitor absorbance with an alternate AUFS setting or to make the primary absorbance measurement using a specific AUFS value on channel A Example While operating in single wavelength mode specifying an AUFS value of 2 0000 on the second channel provides 1 000 V AU on the channel B 2 V output The detector automatically engages the second order filter for all wavelengths 370 nm Operating the detector in dual wavelength mode You can operate the detector with expanded chart out selections using dual wavelength mode In addition to absorbance offered with single wavelength mode dual wavelength mode offers the following functions Absorbance A and B MaxPlot RatioPlot A B Difference plot A B Using the operator interface 3 31 Changing from single to dual wavelength
153. ressure of the tubing Attention Manipulez les tubes en polym re sous pression avec precaution Portez syst matiquement des lunettes de protection lorsque vous vous trouvez proximit de tubes en polym re pressuris s Eteignez toute flamme se trouvant proximit de l instrument Evitez d utiliser des tubes s v rement d form s ou endommag s Evitez d utiliser des tubes non m talliques avec du t trahydrofurane THF ou de l acide sulfurique ou nitrique concentr Sachez que le chlorure de m thyl ne et le dim thylesulfoxyde entrainent le gonflement des tuyaux non m talliques ce qui r duit consid rablement leur pression de rupture Vorsicht Bei der Arbeit mit Polymerschl uchen unter Druck ist besondere Vorsicht angebracht In der N he von unter Druck stehenden Polymerschlauchen stets Schutzbrille tragen Alle offenen Flammen in der N he l schen Keine Schl uche verwenden die stark geknickt oder berbeansprucht sind Nichtmetallische Schl uche nicht f r Tetrahydrofuran THF oder konzentrierte Salpeter oder Schwefels ure verwenden Durch Methylenchlorid und Dimethylsulfoxid k nnen nichtmetallische Schl uche quellen dadurch wird der Berstdruck des Schlauches erheblich reduziert Warnings that apply to all Waters instruments A 7 A A 8 Attenzione fare attenzione quando si utilizzano tubi in materiale polimerico sotto pressione Indossare sempre occhiali da lavoro protettivi nei pressi
154. rol To operate under Empower or MassLynx control the detector uses the Ethernet bus connector see page 2 13 To connect the detector to your HPLC system see page 2 6 When under the control of an external data system the detector is operating under remote control conditions The Remote Control icon incorporating the letter E appears on the absorbance screen see the table on page 3 5 See page 2 10 for more information on connecting the detector to an external system Verifying that the detector operates properly After you have installed the detector verify that it is operating properly by performing the procedures in this section Requirement For complete validation procedures you must obtain the Waters accuracy and linearity cuvette kits and system qualification tool for the detector For spare parts details see the Waters Quality Parts Locator on the Waters Web site s Services amp Support page Tip Before you pump solvent or mobile phase through the system flush the lines with filtered degassed and sparged HPLC grade methanol Then pump your mobile phase provided that there are no miscibility problems at 1 mL min for 15 minutes minimum Before you begin Because the detector is shipped dry you must pump solvent through the unit before its initial use Rule To ensure accurate verification be sure to start up the detector and follow steps 1 to 3 in this section and 1 to 4 in Recording sample and re
155. round such tubing Wear eye protection Extinguish all nearby flames Do not use tubing that is or has been stressed or kinked Do not expose nonmetallic tubing to incompatible compounds like tetrahydrofuran THF and nitric or sulfuric acids Be aware that some compounds like methylene chloride and dimethyl sulfoxide can cause nonmetallic tubing to swell which significantly reduces the pressure at which the tubing can rupture Mass spectrometer flammable solvents warning This warning applies to instruments operated with flammable solvents Warning Where significant quantities of flammable solvents are involved a continuous flow of nitrogen into the ion source is required to prevent possible ignition in that enclosed space Ensure that the nitrogen supply pressure never falls below 690 kPa 6 9 bar 100 psi during an analysis in which flammable solvents are used Also ensure a gas fail connection is connected to the LC system so that the LC solvent flow stops if the nitrogen supply fails Warning symbols A 3 A 4 Mass spectrometer shock hazard This warning applies to all Waters mass spectrometers Warning To avoid electric shock do not remove the mass spectrometer s protective panels The components they cover are not user serviceable This warning applies to certain instruments when they are in Operate mode Warning High voltages can be present at certain external surfaces of the mass spectrometer when the in
156. rror gt in place of the run time absorbance on the absorbance screen See page 5 2 for a list of startup diagnostic test failures error messages and recommended recovery actions See page 5 21 for hardware related causes of startup diagnostic test failure and corrective actions Idle mode When the detector is successfully started it defaults to idle mode see the figure 2489 detector idle mode screen on page 3 4 When it is not performing any function requiring the shutter to be open local methods scans noise test and so on the shutter is closed and the detector remains in idle mode with the lamp lit The Closed Shutter limits unnecessary UV light from reaching the detector s optics bench Initializing the detector 3 3 2489 detector idle mode screen AP Idle Mode gt SX i A x Bue 78 Using the operator interface Using the display 3 4 The detector employs a 128 x 64 bitmap graphic display and a 24 key membrane keypad for the operator interface After the startup diagnostic tests are run successfully the detector displays the absorbance or home screen Detector absorbance screen Absorbance Lamp on off Shift on off Tus Single dual wavelength XT ZA Channel selector AP 0 0 00 1 Ea QM lock unlock Wavelength y Local method Remote control 52 Sensitivity 2 0000 aurs mm Un time minutes Sticky diagnostic test on off Nex
157. rt Using ethanol or methanol clean the contaminated part Blow dry with nitrogen Using the Flow Cell Rebuild Kit replace any flow cell parts that are scratched burred damaged or do not come clean using nitrogen Rebuild the flow cell by following the procedure in the next section Rebuilding the flow cell After cleaning or replacing other flow cell parts rebuild the flow cell To rebuild the flow cell 1 10 11 Using the plastic tweezers remove the new clear plastic gaskets from the Flow Cell Rebuild Kit and inspect them for dirt Drop one clear plastic gasket into the groove at the bottom of the entrance lens end of the flow cell body Inspect the entrance lens and use nitrogen to blow dust off if necessary Using plastic tweezers drop the entrance lens into the flow cell body curved side facing up With the tan colored entrance lens holder curve facing down use the torque wrench to tighten the entrance lens screw into the flow cell body to 0 904 N m 128 in oz or 8 in Ib Flip the flow cell body over to the exit window side Using plastic tweezers inspect the second new gasket for cleanliness Drop the clear plastic gasket into the groove at the bottom of the cuvette lens end of the flow cell body Inspect the exit window for cleanliness Using nitrogen clean the exit window if necessary Using plastic tweezers place the exit window into the flow cell body With the tan colored exit win
158. s The software includes fast or normal filtering constants at each data rate that are appropriate for high speed or high sensitivity applications respectively The following figure shows the relationship between increased filter time constant and absorbance Filter time constant comparison 0 6 0 5 0 4 0 3 0 2 Absorbance 0 1 0 0 2 0 4 0 6 0 8 1 1 2 1 4 Time minutes Tip Although the peak shape shows some distortion and the signal output is delayed with different time constants the peak area remains the same Theory and Principles of Operation Wavelength verification and test The detector s deuterium arc lamp and integral erbium filter exhibit peaks in the transmission spectrum at known wavelengths Upon startup the detector verifies calibration by comparing the locations of these peaks with expected wavelengths based on calibration data stored in the detector s memory If the results of this verification differ from the stored calibration by more than 1 0 nm the detector displays a Wavelength Verification Failure message The detector verifies rather than recalibrates on startup to avoid errors arising from residual materials left in the flow cell and or the cuvette Requirement Always ensure that the cuvette is removed and the cuvette holder and front left hand panel door are secured during startup verification You can initiate a manual wavelength calibration at any time A manual cal
159. shold events within the displayed method Tip If you change to a timed or threshold event within a method the asterisk appears Method indicating that the method 1s no longer the same as the stored method you retrieved in step 1 You can then store the method containing the altered event s in the same storage slot 3 40 Preparing the Detector Resetting a method Resetting a stored method is a two step process First you reset the current conditions to the defaults Then you save the defaults in one of the storage locations See page 3 18 for the parameter default settings To clear one or more methods 1 2 Return to the Method choice list by pressing METHOD Shift A B Press 5 Reset method Result A message appears asking whether you approve of setting current conditions to factory defaults Tip If you press Enter these events take place All timed events are deleted All threshold events are disabled All other operating parameters of the method A AUFS etc are set to defaults If you press Cancel Shift 0 the display returns to the Method choice list Recommendation To prevent loss of the current conditions store them in one of the available storage slots before you clear the method After clearing the storage slots you can restore the previous conditions Press 4 Store method and enter a storage location number Tip To clear other stored methods repeat this step until you cle
160. sses good operating procedures and the techniques necessary to validate system and method performance Safety advisories Consult Appendix A for a comprehensive list of warning and caution advisories Operating this instrument When operating this instrument follow standard quality control QC procedures and the guidelines presented in this section Applicable symbols Symbol Definition Confirms that a manufactured product complies with all applicable European Community directives e ABN 49 065 444 751 Australia C Tick EMC Compliant with all applicable United States and Canadian ij Confirms that a manufactured product complies i safety requirements se of CAN CSA C22 2 No 61010 1 second edition including Amendment 1 or a later version of the same standard incorporating the same level of testing requirements C This product has been tested to the requirements l Audience and purpose This guide is intended for personnel who install operate and maintain 2489 UV Visible detectors Intended use of the 2489 UV Visible detector Waters designed the 2489 UV Visible detector to analyze and monitor many compounds Calibrating To calibrate LC systems follow acceptable calibration methods using at least five standards to generate a standard curve The concentration range for standards should include the entire range of QC samples typical specimens and atypical specimens When
161. ssing SCAN Shift Chart mark Press 2 Store last scan Tip When you select Store last scan you are storing the zero scan and the sample scan as a pair Storage slot number box In the slot number box enter a number from 1 to 3 Press Enter to store the last sample scan paired with its zero scan Getting information about a stored spectrum To obtain information about a stored spectrum 1 2 Press SCAN Shift Chart mark to view the Scan choice list Press 3 Get scan info Result A slot number box appears with a default of Last the most recently stored spectrum Press Enter to get information about the last stored spectrum or type the number 1 to 3 of the stored spectrum for which you want information and then press Enter Result A screen containing the following information appears Storage slot number of the selected scan or Last scan 2 range Displays the wavelength range for the selected spectrum Pace Displays the pace for the selected spectrum Press Enter to exit the information screen and return to the Scan choice list 3 54 Preparing the Detector Reviewing a stored spectrum Once you store a spectrum you can retrieve it for review from one of the five available storage slots by selecting the Review option from the Scan choice list To review a spectrum 1 Press SCAN Shift Chart mark to view the Scan choice list 2 Press 4 Review Tip When you select Review
162. stalled To power on the detector press the On Off switch located on the front lower right corner of the unit At startup the detector beeps three times and runs a series of startup diagnostic tests If it passes all startup diagnostic tests the Startup Diagnostics screen appears with OK following each test Startup Diagnostics screen STARTUP DIAGNOSTICS TPU E Rom g sci I3 RAM ey cPiB iW Lop Ei asp A cpu fg After it displays the Startup Diagnostics screen the detector displays the following series of messages in sequence a process that takes about five minutes Initializing grating Initializing system Preparing the Detector Lighting lamp Warmup time left n minutes Homing optical filter Searching for 656 nm Optimizing system performance Finding calibration peaks Restoring last setup Completing initialization When initialization ends the detector displays the absorbance screen shown see page 3 4 The sections Using the keypad on page 3 7 and Navigating the user interface on page 3 12 provide more information on this and subsequent screens Tip For normal use allow the detector to warm up for at least 30 minutes before operating Diagnostic test failure If one or more of the internal startup diagnostic tests show a failing result the detector beeps and displays an error message For serious errors it displays the word Error in brackets lt E
163. strument is in Operate mode To avoid non lethal electric shock make sure the instrument is in Standby mode before touching areas marked with this high voltage warning symbol Biohazard warning This warning applies to Waters instruments that can be used to process material that might contain biohazards substances that contain biological agents capable of producing harmful effects in humans Warning Waters instruments and software can be used to analyze or process potentially infectious human sourced products inactivated microorganisms and other biological materials To avoid infection with these agents assume that all biological fluids are infectious observe Good Laboratory Practices and consult your organization s biohazard safety representative regarding their proper use and handling Specific precautions appear in the latest edition of the US National Institutes of Health NIH publication Biosafety in Microbiological and Biomedical Laboratories BMBL Safety Advisories Chemical hazard warning This warning applies to Waters instruments that can process corrosive toxic flammable or other types of hazardous material A A Warning Waters instruments can be used to analyze or process potentially hazardous substances To avoid injury with any of these materials familiarize yourself with the materials and their hazards observe Good Laboratory Practices GLP and consult your organization s safety representative regard
164. sts are active the wrench icon does not appear on the absorbance screen When you power off the detector sticky diagnostic tests become inactive Absorbance screen with sticky diagnostic tests active vtt AP 0 0002 EN p Wrench icon A 5 ench ic Ee Z 20000 aurs The user selectable sticky diagnostic tests are Fix Set Voltage Output Fix Set Absorbance Input Generate Test Peaks A Optical Filter Override Caution Application of sticky diagnostic tests affects results To clear changes to the voltage output or absorbance input or to make a manual optical filter change select 1 Reset instrument from the Diagnostics choice list or cycle power to the detector The table below lists diagnostic tests by their number on the choice list see page 5 11 for more information Detector diagnostic tests Diagnostic test Description 1 Reset instrument Resets all diagnostic tests to defaults Renders sticky diagnostic tests inactive and removes the wrench icon 2 Sample amp ref energy Makes it possible to view sample and reference energy displayed in nanoamps on channel A or B 5 10 Error Messages Diagnostic Tests and Troubleshooting Detector diagnostic tests Continued Diagnostic test 3 Input amp output gt gt Description List of diagnostic tests to control four contact closure inputs and two switch outputs Auto zero offset Fix absorbance
165. t You can recall the absorbance screen at any time by pressing the HOME key When you first use of the detector the absorbance screen shows the factory set defaults Afterward the absorbance screen shows the settings displayed before the detector was last powered off The absorbance screen continues to change as the run continues The detector monitors absorbance of one or two wavelengths in real time while allowing you to modify all the parameters discussed in the table below You can use the A B key to toggle between absorbance screens for channels A and B Preparing the Detector Absorbance and message icons The absorbance screens and message screens in the detector program display the icons or fields shown in the figure on page 3 4 and described in the table below For a list of ranges and defaults for the function icons and fields described in this table see the table titled Primary and secondary function method parameters on page 3 18 Caution Changing the sensitivity AUFS setting affects the 2 V output For example 1 AU gives 0 5 AU V and 2 AU gives 1 AU V Absorbance and message screen icons Icon or field Icon field name Function Field Sensitivity or Selects the chart sensitivity in requiring AUFS absorbance units full scale AUFS for entry the selected channel Ethernet signal is not affected Field Wavelength Selects the wavelength monitored on the requiring selecte
166. t miscible in water first use an intermediary solvent Resume pumping mobile phase Reattach the column Tip Waters recommends renormalizing in 100 water before resuming analyses If the flow cell remains dirty or blocked reverse flush it Removing and cleaning the flow cell If flushing the flow cell is not effective follow the procedures to remove the flow cell and inspect for dirty or broken windows or dirty gaskets Clean and replace parts if necessary Before removing the flow cell assembly purge the flow cell with nitrogen and allow it to dry To purge the flow cell 1 Connect the nitrogen supply line to the sample inlet Route the sample line to waste Purge the cell for 25 to 30 minutes at 103 to 138 kPa 1 to 1 4 bar 15 to 20 psi Allow the flow cell to dry completely Disconnect the detector inlet outlet tubing from the main column connection and cap Maintaining the flow cell 4 7 Disassembling and reassembling the flow cell Before you begin Take the following precautions when disassembling and reassembling the flow cell To prevent contamination use powder free finger cots or gloves when touching the flow cell lenses or window Take care to prevent scratching of the flow cell parts Usea clean nonparticulating cloth or a similar clean surface in the work area where you are removing rebuilding and replacing the flow cell Caution Always replace the flow cell gaskets when cleaning
167. tain functional groups found in most compounds absorb light selectively These groups known as chromophores and their behavior can be used to categorize the detection of sample molecules Wavelength selection C 11 The table below lists some common chromophores and their detection wavelengths Ajax as well as the molar absorptivity Emax of each group Use this information as a guide to select the optimal operating wavelength for a particular analysis Because of the diversity possible within a given sample scanning over a range of wavelengths may be necessary to determine the best wavelength for a particular analysis Electronic absorption bands of representative chromophores Chromophore ENEMIES max IUE Amax pes configuration nm L m cm nm L m cm Ether 0 185 1000 Thioether 8 194 4600 215 1600 Amine NH2 195 2800 Thiol SH 195 1400 Disulfide S S 194 5500 255 400 Bromide Br 208 300 Todide I 260 400 Nitrile C N 160 Acetylide CaC 175 180 6000 Sulfone SO2 180 Oxime NOH 190 5000 Azido gt C N 190 5000 Ethylene C C 190 8000 Ketone 2 0 195 1000 270 285 18 30 Thioketone 20 28 205 strong Esters COOR 205 50 Aldehyde CHO 210 strong 280 300 11 18 Carboxyl COOH 200 210 50 70 Sulfoxide 28250 210 1500 Nitro NO9 210 strong Nitrile ONO 220
168. tes and digitizes the currents from the photodiodes for processing by the signal processing electronics and output to a computer chart recorder or integrator Waters TaperSlit Flow Cell The Waters TaperSlit Flow Cell used in this detector renders the detector baseline less sensitive to changes in mobile phase refractive index RI RI changes occur during gradient separations or result from temperature or pump induced pressure fluctuations To achieve RI immunity a combination of a spherical mirror a lens at the entrance of the flow cell and a taper to the internal bore of the flow cell prevents light rays from striking the internal walls of the flow cell An additional feature of the TaperSlit flow cell and the reason for its name is the shape of the flow cell entrance which matches the shape of the entrance slit The detector achieves higher light throughput for a given spectral resolution via the TaperSlit cell design compared to a conventional flow cell with a circular entrance As shown in the figure below in a conventional cell light bends and hits the wall of the flow cell Four beams go in but only two come out In the Waters TaperSlit analytical cell the combination of the lens and TaperSlit bore geometry prevents light from hitting the cell walls Four beams go in and four beams come out Theory and Principles of Operation Comparison of flow cell characteristics _ wT Conventional cell FU Window UV light
169. the Empower software or a MassLynx system you must select a data sampling rate of 1 point per second 3 22 Preparing the Detector Configuring event inputs contact closures You use the CONFIGURE key also to edit event input settings and specify switched output settings Using the Enter key and the numeric keypad or the s and t keys to select the appropriate entry you can edit four fields on the second Configuration screen Tip The default for Inject Chart mark and Auto Zero 1s Low the default for Lamp is Ignore Inject You can specify the inject input to signal the start of a run This event resets the run time clock and applies initial method conditions immediately A High Start run when contact closure changes from off open to on closed Low Start run when contact closure changes from on closed to off open Ignore Do not respond to inject start input Chart mark You can specify the chart mark input to create a chart mark on channel A and or channel B You determine the response of the channel using the enable chart mark function explained in the table on page 3 18 and shown in the figure on page 3 18 High Create chart mark s when contact closure changes from off open to on closed Low Create chart mark s when contact closure changes from on closed to off open Ignore Do not respond to chart mark input Auto zero You can configure the auto zer
170. the composition of mixed solvents Vacuum degassing The in line vacuum degasser operates on the principle of Henry s Law to remove dissolved gases from the solvent Henry s Law states that the mole fraction of a gas dissolved in liquid is proportional to the partial pressure of that gas in the vapor phase above the liquid If the partial pressure of a gas on the surface of the liquid 1s reduced for example by evacuation then a proportional amount of that gas comes out of solution C 8 Solvent Considerations Vacuum degassing can change the composition of mixed solvents Solvent degassing considerations Select the most efficient degassing operation for your application To remove dissolved gas quickly consider the following Sparging Helium sparging gives stable baselines and better sensitivity than sonication in a detector and prevents reabsorption of atmospheric gases Use this method to retard oxidation when you are using tetrahydrofuran or other peroxide forming solvents Vacuum degassing The longer the solvent is exposed to the vacuum the more dissolved gases are removed Two factors affect the amount of time the solvent is exposed to the vacuum Flow rate At low flow rates most of the dissolved gas is removed as the solvent passes through the vacuum chamber At higher flow rates lesser amounts of gas per unit volume of solvent are removed Surface area of the degassing membrane The length of the degassin
171. ting to baseline maintains the previous baseline level when the wavelength change is made The default is to zero Using the operator interface 3 17 Secondary functions of the absorbance screen AS ECH 2 0000 sues es Absorbance screen HOME Press Next AP analog adi D Hz i Normal B Filter time constant 2 0000 i Sec ae Analog rate and filter time constant available for both and 1A Press Next do absorbance A out absorbance A Absorbance offset and auto zero on inject available for both and 12 Press Next _ Agen e offset mV chart A polarity L E Voltage offset and chart polarity available for both and AA Press Next AP min imum 01008 minimum rate maximum 5of5 Minimum AU minimum ration and maximum ration available only for 1 4of5 Primary and secondary function method parameters Function Screen Type A 1 Numeric Wavelength Absorbance screen AUFS 1 Numeric 3 18 Preparing the Detector Units Range Integer 190 nm to 700 nm nm AUFS 0 0001 to 4 0000 Default 254nm 2 0000 Primary and secondary function method parameters Continued Function Screen Type Units Range Default Analog rate 2 of 4 or Choice Hz A 10 20 40 80 10 2 of 5 AA
172. tor through the B inputs and outputs terminal on the rear panel of the detector as shown in the figure below To connect the detector to the e SAT IN module 1 Using the electrical insulation stripping tool strip about 3 mm 1 8 inch from one end of the e SAT IN 9 pin connector exposing the white and black wires Connecting to other devices 2 21 2 Connect the other end of the cable to the Channel 1 or Channel 2 connectors on the front panel of the e SAT IN module 3 For Channel 1 connect the white wire to pin 1 on I Analog 1 connect the black wire to pin 3 on I Ground e SAT IN module channel 1 connection to detector 2489 connector 1 Analog 1 2 Analog 1 3 Ground 4 Analog 2 5 Analog 2 6 Switch 1 7 Switch 1 8 Ground 9 Switch 2 10 Switch 2 4 For Channel 2 connect the white wire to pin 4 on I Analog 2 connect the black wire to pin 8 on I Ground 2 22 Installing the Detector e SAT IN module channel 2 connection to detector 2489 connector Analog 1 Analog 1 Ground Analog 2 1 2 3 4 5 Analog 2 Waters e SAT IN zle 6 Switch 1 i 7 8 9 1 Switch 1 Ground Switch 2 O Switch 2 CHANNEL1 CHAMBIEL 2 5 Configure the serial port for the e SAT IN module as described in the Empower 2 Installation and Configurati
173. trical power connections 2 9 provide a traditional unattenuated dedicated 1 V AU output because its enhanced working range is above 2 AU The analog output range specifications are 0 1 V to 2 1 V Youcan set the AUFS value individually for the output on each channel Volts per AU are calculated as follows Volts out Absorbance x 2V AUFS Example An AUFS setting of 2 0000 provides a traditional 1 V AU output An AUFS setting of 4 0000 provides a 0 5 V AU output which supports chromatography above 2 AU Switched outputs You can program two switch contact closures to turn on turn off toggle pulse once for a defined duration or pulse repetitively for a specified period of time Event inputs Four general purpose TTL contact closures on the detector A inputs terminal support the following functions Remote or inject start Lamp on off Chart mark input Auto zero Ethernet interface The Ethernet connection on the rear panel of the detector provides remote control operation and direct data acquisition from Waters Empower and MassLynx workstations Making signal connections 2 10 The rear panel of the detector see the figure on page 2 9 provides two analog connectors and an Ethernet communications port for operating the detector with external devices Make the signal connections to your detector considering the following conditions Operating mode you select for t
174. vents in use Warning Avoid electric shock Do not open the detector s cover The components within are not user serviceable Power off and unplug the detector before performing any maintenance operation on the instrument Caution To avoid damaging electrical parts never disconnect an electrical assembly while power is applied to the detector To completely interrupt power set the module s power switch to off and then unplug the power cord from the AC outlet Wait 10 seconds before you disconnect an assembly Spare parts Replace only parts mentioned in this document For spare parts details see the Waters Quality Parts Locator on the Waters Web site s Services amp Support page Maintenance considerations 4 3 Proper operating procedures Removing the front left panel cover Caution There are no user serviceable parts inside the detector Do not remove the top cover To maintain optimum performance replace the front left panel cover before resuming normal operation of the detector To remove the front left panel cover 1 Holding on to the top of the cover gently pull the bottom away from the unit 2 Gently remove the top of the cover and store it nearby The following figure illustrates the detector with the front left hand panel cover removed Detector with front left front panel cover removed 4 4 Maintaining the Detector Routine maintenance The detector requires minimal rout
175. with a 2695 Separations Module Empower or MassLynx software or a non Waters product Diagnostic tests The detector performs some user selected diagnostic tests to help you troubleshoot basic system problems See page 5 11 for diagnostic descriptions and instructions on how to use them Error messages that can appear as you start up or operate the detector and suggested corrective actions are described in the tables on page 5 3 and page 5 6 5 20 Error Messages Diagnostic Tests and Troubleshooting Power surges Power surges line spikes and transient energy sources can adversely affect detector operations Be sure that the electrical supply used for the detector is properly grounded and free from any of these conditions Hardware troubleshooting This section contains general hardware troubleshooting for the detector General system troubleshooting energy error on startup installed or a UV absorber in flow cell Symptom Possible cause Corrective action Analog output AUFS setting Reset the AUFS setting incorrect changed Calibration or Cuvette is 1 Remove the cuvette 2 Flush the flow cell 3 Perform manual calibration Detector inoperative Fuse blown Confirm the front panel display is operational if not replace the AC rear panel fuse No power at outlet Test the outlet by connecting another electrical unit known to be in working order and determine whether it operates Deut
176. without tick marks AUFS The sensitivity setting used for scaling the charted spectrum Scan of erbium standard in a cuvette from 190 nm to 600 nm at 200 nm min with tick marks every 20 nm 0 6 0 5 04 3 03 0 2 is i 0 0 W LA U 290 390 490 Wavelength nm Scan of erbium standard in a cuvette from 190 nm to 600 nm at 200 nm min without tick marks 0 6 0 5 0 4 0 3 0 2 M ln 0 0 ZA 290 390 490 Wavelength nm AU Specify these parameter values when you select a scan zero or sample 3 44 Preparing the Detector When you select a zero scan the detector displays three additional screens labeled 2 of 4 3 of 4 and 4 of 4 You can change all parameters on these screens including starting and ending wavelength and the pace parameter When you select a sample scan the detector displays two additional screens labeled 2 of 3 and 3 of 3 see the figure on page 3 49 You cannot change the starting and ending wavelength values or the pace parameter When you run a zero scan you establish starting and ending wavelengths pace tick marks and sensitivity for both the zero scan and subsequent sample scans Run sample scans within 15 minutes of the baseline zero scan The most recent zero scan performed or retrieved remains current until you perform or retrieve another scan The zero scan should be appropriate for the subsequent sample scans you perform The sample scan adopts the starting and ending wavelengths and
177. you actually are retrieving the zero scan and the sample scan that were stored as a pair 3 Enter the storage slot number 1 to 3 of the spectrum you want to review 4 Press Enter The message Retrieving spectrum n appears Once you retrieve a spectrum for review you can view it graphically and adjust the wavelength and AU ranges if necessary You can also run a new sample scan based on the retrieved zero scan Subtracting a spectrum Once you store more than one spectrum you can create a difference spectrum Tip The current spectrum is the spectrum that the stored spectrum whose slot number you enter is subtracted from To subtract and review the difference spectrum Rule To subtract the stored spectrum from the current spectrum the starting and ending wavelengths ol and 42 and the pace of both spectra must be identical 1 Press SCAN Shift Chart mark 2 Press 5 Subtract amp review 3 Enter the storage slot number 1 to 3 of the spectrum you want to subtract out from the current or retrieved spectrum 4 Press Enter Once the difference spectrum appears you can store the results in one of the three storage slots Scanning spectra 3 55 Replaying a spectrum You can play back the current spectrum or a stored spectrum in real time using the Real time replay function on the Scan choice list The detector plays back the selected spectrum in real time both on the detector display and out the analog c
178. you can see liquid through the holder s aperture That is the liquid must completely cover the aperture Because the cuvette holder is angled use your thumb or forefinger to ensure the cuvette 1s secure in the slot and does not slide forward Ensure that it does not become dislodged when you are replacing the cuvette holder 4 Gently guide the cuvette holder back into the flow cell assembly until it bottoms in the space 3 58 Preparing the Detector Replacing the cuvette holder 5 Refit the panel Rules To prevent invalid chromatographic results remove the cuvette from the detector and replace the empty holder after running cuvette scans To maintain optimum system performance refit the front left panel cover before resuming normal operation of the detector 6 Insert a reference cuvette containing the mobile phase standard and run a zero scan 7 Replace the reference cuvette with a cuvette containing your analyte dissolved in mobile phase solvent and run a sample scan 8 Use the storage review subtract and review and replay functions to analyze the data obtained Scanning using a flow cell and a syringe If you do not have a cuvette you can scan using a manually filled flow cell Requirement Before you scan using a flow cell make sure no cuvette is in the cuvette holder Scanning spectra 3 59 To run a spectrum using a flow cell 1 With a syringe fill the flow cell with
179. you store spectra and acquire new spectra for subtraction beware the differences if any in flow cell conditions Ideally you perform both the zero and sample scans using the cuvette when the HPLC instruments are in the idle or static state under identical flow cell conditions Caution Handle the cuvette gently and on the frosted sides only Fingerprints on the clear quartz interfere with the light path and compromise the integrity of cuvette scanning operations Before you begin Recommendation To ensure accurate results use a 10 mm path length quartz cuvette and matched pairs from the same manufacturing lot of quartz cuvettes for your zero and sample scans Before beginning a scan using the cuvette 1 Flush the flow cell with the eluent you are going to scan 2 Wipe the clear portion of the cuvette with low lint nonabrasive tissue wipes Scanning spectra 3 57 Cuvette scanning procedure To begin a cuvette scan 1 Remove the detector s front left hand panel cover 2 Remove the cuvette holder sliding it toward you Removing the cuvette holder 3 With the spring guide facing you gently insert the cuvette containing eluent up under the guide with the cap facing upward into the holder and a frosted side of the cuvette facing up See the figure on page 3 57 Recommendations e Ensure that you have enough liquid 8 mL in the cuvette so that when it is inserted into the holder

Waters 2489 UV/Visible Detector Operator`s Guide

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