Waters 2489 Operator'S Manual Download Page 20

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1-4

Theory and Principles of Operation

Principles of operation

To use the detector effectively, you should be 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 the following:

•

High brightness deuterium (D

) lamp

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Two mirrors: one off-axis ellipsoidal mirror and one spherical 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

Summary of Contents for 2489

Page 2: ...shall Waters Corporation be liable for incidental or consequential damages in connection with or arising from its use Trademarks Waters is a registered trademark of Waters Corporation Empower e SAT IN...

Page 3: ...s via the Internet telephone or conventional mail Waters contact information Contacting medium Information Internet The Waters Web site includes phone numbers for Waters locations worldwide Go to www...

Page 4: ...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...

Page 5: ...r follow acceptable calibration methods using at least five standards to generate a standard curve The concentration range for standards should cover the entire range of quality control samples typica...

Page 6: ...rily When analyzing samples from a complex matrix such as soil tissue serum plasma whole blood and so on note that the matrix components can adversely affect results To minimize these matrix effects W...

Page 7: ...ith the Medicines and Healthcare Products Regulatory Agency MHRA at Market Towers 1 Nine Elms Lane London SW8 5NQ The reference number is IVD000167 Waters Corporation Micromass UK Ltd Floats Road Wyth...

Page 8: ...viii...

Page 9: ...al modes 1 11 Single wavelength mode 1 11 Primary parameters 1 11 Secondary parameters 1 12 Dual wavelength mode 1 12 Chart Out selection modes 1 13 Spectrum scanning 1 13 Cuvette operations 1 14 Rati...

Page 10: ...erating Chart Mark on Inject 2 19 Connecting to other devices 2 20 Required materials 2 21 Connecting the cables 2 21 Connecting the detector to Empower using an e SAT IN module 2 21 e SAT IN module 2...

Page 11: ...ary and secondary functions 3 14 Operating the Trace and Scale functions 3 20 Operating other detector functions 3 22 Configuring the detector 3 22 Configuring event inputs contact closures 3 23 Setti...

Page 12: ...a method 3 42 Clearing events 3 43 Scanning spectra 3 44 Before you begin 3 44 Scanning new spectra 3 48 Zero scan 3 49 Running the sample scan 3 52 Storing a spectrum 3 56 Getting information about...

Page 13: ...6 Before you begin 4 6 Tools required 4 7 Removing the flow cell assembly 4 7 Disassembling the flow cell 4 10 Inspecting cleaning and replacing damaged flow cell components 4 13 Rebuilding the flow...

Page 14: ...amp display and keypad diagnostic tests 5 15 Using the Change Lamp diagnostic test 5 15 Using the other detector diagnostic tests 5 17 Service diagnostic tests 5 19 Troubleshooting 5 19 When you conta...

Page 15: ...rSlit Flow Cell specifications B 5 C Spare Parts C 1 D Solvent Considerations D 1 Introduction D 2 Clean solvents D 2 Solvent quality D 2 Preparation checklist D 2 Water D 2 Using buffers D 2 Tetrahyd...

Page 16: ...D 8 Sparging D 8 Vacuum degassing D 8 Solvent degassing considerations D 8 Sparging D 8 Vacuum Degassing D 9 Wavelength selection D 9 UV cutoffs for common solvents D 9 Mixed mobile phases D 10 Wavel...

Page 17: ...atures and describes the theory and principles of operation See also Appendix B for system specifications and Appendix D for information on high performance liquid chromatography HPLC solvent consider...

Page 18: ...stand alone unit with a chart recorder or integrator or as an integral part of a Waters chromatography system The detector can be configured with Empower or MassLynx software systems Features The dete...

Page 19: ...sample analysis Allows the spectrum of any sample placed in the cuvette to be recorded Method editing and storage Supports basic method programming storage and retrieval from the front panel Full dia...

Page 20: ...cell Electronics Detector optics The Waters 2489 UV Visible Detector optics are based on a Fastie Ebert monochromator and include the following High brightness deuterium D2 lamp Two mirrors one off a...

Page 21: ...rsed 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...

Page 22: ...nce 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 re...

Page 23: ...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 selec...

Page 24: ...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 baseli...

Page 25: ...ion 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...

Page 26: ...n algorithm replaces the stored data with the new data The detector wavelength verification procedures establish an approximate Home position using a grating homing sensor Once Home is established the...

Page 27: ...s 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 paramete...

Page 28: ...xplain 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 fr...

Page 29: ...AUFS setting This mode is useful when looking at multiple compounds with absorbancies at two separate wavelengths with one data channel RatioPlot A B This mode produces the ratio of absorbance from tw...

Page 30: ...hat 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 59 and Scanning using a flow...

Page 31: ...hannel For an explanation of the RatioPlot procedure see the discussion Obtaining a RatioPlot on page 3 34 MaxPlot The MaxPlot function monitors absorbance at two selected wavelengths and plots the ma...

Page 32: ...1 16 Theory and Principles of Operation...

Page 33: ...ard 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 insta...

Page 34: ...f applicable on file Verification ensures proper operation of the detector optics and electronics For proper verification procedures see Verifying the detector on page 3 27 Start installation procedur...

Page 35: ...Caution Do not access the instrument through the top cover Access the instrument through the front left panel where the lamp housing flow cell assembly and cuvette holder are located TP02804 28 4 cm...

Page 36: ...the flow cell Installation site requirements Parameter Requirement Operating temperature range 4 to 40 C 39 to 104 F Storage temperature range 30 to 60 C Relative humidity 20 to 95 noncondensing Stor...

Page 37: ...contains the following items Waters 2489 UV Visible Detector Startup Kit which includes this guide Power cord Release notes Warning To avoid electric shock use the SVT type power cord in the United St...

Page 38: ...Technical Service Representative or call Waters corporate headquarters for assistance at 1 508 478 2000 U S Tip Make sure the instrument serial number found on the nameplate on the rear panel or insi...

Page 39: ...ing columns The fluid 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...

Page 40: ...id line connections 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 taper end facing the end of...

Page 41: ...revent reabsorption of dissolved oxygen for systems using vacuum degassers Waters recommends that you run the solvent degasser continuously when operating the detector at wavelengths less than 230 nm...

Page 42: ...input output voltage current specifications see the Table titled Operational specifications on page B 2 The 2 V output for I and II is scaled based on the AUFS absorbance units full scale setting for...

Page 43: ...ernet connection on the rear panel of the detector allows remote control and direct data acquisition from Waters Empower and MassLynx workstations Making signal connections The rear panel of the detec...

Page 44: ...ectors 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 Yes Signal connections complete No Yes Connect to other d...

Page 45: ...ed events Defines the start of a run typically an injection and resets and starts the run time clock to 0 00 minutes Initial conditions apply immediately Lamp On Off a Configurable input to allow an e...

Page 46: ...th mode you must select a data rate of 1 point per second in the data system method editor In Empower software the data rate parameters defaults to 1 point per second Analog 2b 2 V full scale analog o...

Page 47: ...boratory acquisition and control environment or LAC E 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 detector rea...

Page 48: ...ector 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 config...

Page 49: ...by making the connections shown in the table and figure below Separations module connections to the detector for turning the lamp on or off Detector connections to separation module lamp on or off 269...

Page 50: ...tion make the connections summarized in the table and illustrated in the figure below Before you can generate an auto zero from the separations module you need to configure the auto zero signal at the...

Page 51: ...illustrated in the figure below Before you can generate a chart mark from the separations module you need to configure the chart mark signal at the detector front panel The default chart mark signal...

Page 52: ...s This section describes how to connect the detector to the following devices e SAT IN module instead of Ethernet Waters 745 745B 746 Data Module Chart recorder Waters 600 Series Pump Waters 717plus A...

Page 53: ...iate connector 5 Tighten the screw until the wire is held securely in place 6 Reinsert the terminal 7 Press firmly to ensure that it is inserted fully Connecting the detector to Empower using an e SAT...

Page 54: ...h To prevent damage to the module 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 prop...

Page 55: ...ol strip off about 3 mm 1 8 inch from one end of the e SAT IN 9 pin connector exposing the white and black wires 2 Connect the other end of the cable to either the Channel 1 or Channel 2 connectors on...

Page 56: ...cribed in the Empower 2 Installation and Configuration Guide The table below summarizes the detector connections to the e SAT IN module Detector connections to a e SAT IN module 2489 Detector I e SAT...

Page 57: ...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 To minimize the chance of creating a ground loo...

Page 58: ...2 26 Installing the Detector Data module connections to detector channels A and B Waters 2489 I TP01485 Waters 2489 I TP01486...

Page 59: ...ed in the figure below 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 ot...

Page 60: ...ns on the detector Chart marks You can generate a chart mark from the front panel of the detector A chart mark signal is generated whenever one of the following occurs You press the Chart Mark key on...

Page 61: ...in Making fluid line 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 detector front panel You mu...

Page 62: ...2 Program the pump to provide a pulse output on the applicable switch S1 S2 or S4 at the beginning of each run Refer to the Waters 600E Multisolvent Delivery System User s Guide Section 5 1 2 for mor...

Page 63: ...gure below 2 Program the pump to provide a pulse output on the selected switch at the beginning of each run Refer to the Waters 600E Multisolvent Delivery System User s Guide for more details Pump and...

Page 64: ...tector 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 provid...

Page 65: ...act closure signal to command the detector to perform an auto zero at the start of an injection pump and detector Inject Start connections Waters 2489 Detector II Waters 600 Series Pump Terminal Pin 1...

Page 66: ...ilable pair of Inject Start terminals on the autosampler Autosampler Auto Zero connections to the detector Autosampler and detector Auto Zero connections 2489 Detector A Inputs 717plus Autosampler Ter...

Page 67: ...ctions described in the table and illustrated in the figure below Use any available pair of Inject Start terminals on the autosampler Detector Inject Start connections Autosampler and detector Inject...

Page 68: ...k event input each time a tube is changed at the fraction collector The table below indicates the correct detector to fraction collector and autoinjector to fraction collector connections See also Ref...

Page 69: ...Empower system to control and collect digital data To configure the detector with this system follow the instructions in the Empower online Help to specify parameters and control the detector As part...

Page 70: ...llowing display of the Startup Diagnostics screen the detector displays the following series of messages in sequence for about five minutes Initializing grating Initializing system Lighting lamp Warmu...

Page 71: ...related causes of startup diagnostic test failure and corrective actions Using the operator interface Using the display The detector employs a 128 64 bitmap graphic display and a 24 key membrane keyp...

Page 72: ...secondary function method parameters on page 3 19 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 scr...

Page 73: ...tor operating in single wavelength mode Detector operating in dual wavelength mode Keypad Lock Unlock Open lock Unrestricted keypad entry Closed lock Parameter changes not allowed Sticky Diagnostic ic...

Page 74: ...tem or other control device over the Ethernet bus it displays a cursive m and the current method number or an asterisk that indicates current conditions are not stored as a method Remote control Ether...

Page 75: ...er fields and to press Enter for the functions to take effect Keys that appear in all caps HOME DIAG TRACE METHOD CONFIGURE and SCAN take you directly to a function from most screens For numerical ent...

Page 76: ...nnels SCAN Displays the list of options for generating and manipulating spectra Auto Zero Sets the absorbance offset so that the analog output A and or B depending on the current settings reads 0 AU Y...

Page 77: ...key is active the NEXT arrow appears in the lower right corner of the display Previous When the Next key is available Previous navigates through the screens in the reverse order A B On screens that ha...

Page 78: ...ys 1 See 0 9 above Lamp Displays the lamp use statistics for the currently installed lamp and allows you to turn the lamp on or off The current state of the lamp is indicated by an icon on the absorba...

Page 79: ...active field CE Clears an editing change and returns the contents of a field to its previous value Sets the value to a unique word for some fields For example in the voltage offset diagnostic display...

Page 80: ...s the corresponding number key to select an item immediately Use the up and down arrow keys to scroll through the list then press Enter Tip If you know the number corresponding to the desired choice y...

Page 81: ...screen and select a wavelength mode or you are ready to set up the detector for a run In addition to wavelength mode you must program the following parameters before beginning a run Operating waveleng...

Page 82: ...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 single You can char...

Page 83: ...channel B is larger Volts out Larger absorbance A or B 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 chro...

Page 84: ...f the two absorbances measured on the A and B wavelengths The detector uses the AUFS absorbance offset and voltage offset of the selected channel for scaling Volts out Absorbance difference A B 2 V AU...

Page 85: ...umerical key to clear this box for either or both channels Auto zero on changes This parameter results in an auto zero each time a wavelength change is requested If you disable this function significa...

Page 86: ...ratio and maximum ratio available only for Analog rate and filter tie constant available for both and Absorbance offset and auto zero on inject available for both and Absorbance screen HOME Voltage o...

Page 87: ...er 0 0125 to 5 0 0 5 to 5 0 0 to disable filtering Normal for Other 1 000 Data out single 3 of 4 Choice None Absorbance A Absorbance A Data out dual 3 of 5 Choice None Absorbance A Absorbance B Maxplo...

Page 88: ...isplay 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 AU1 starting or low absorbance The default is auto 3 P...

Page 89: ...jections 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 The figure below shows a...

Page 90: ...ctions Configuring the detector To change default configurations use the Configuration screens Press the CONFIGURE key Shift DIAG The first of two Configuration screens appears see figure below Tip Ot...

Page 91: ...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 chann...

Page 92: ...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 wid...

Page 93: ...screen Use the and 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 v...

Page 94: ...ers 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 de...

Page 95: ...present on the absorbance screen see the table on page 3 4 See Making signal connections on page 2 11 for more information on connecting the detector to an external system Verifying the detector Afte...

Page 96: ...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 Refer to Chapter...

Page 97: ...at 1 mL min for 15 minutes minimum to clean the cell You are now ready to run the peak response verification test If the following test fails repeat it Make the fluid connections described in Making f...

Page 98: ...s Calibrate Shift 3 from the detector keypad A message appears asking whether you removed the cuvette and flushed the flow cell with a transparent solvent Waters recommends methanol or water Cuvette w...

Page 99: ...screen before you recalibrated the detector disappears 4 If calibration is not successful retry recycle power to the detector or see Chapter 5 Operating the detector in single wavelength mode The dete...

Page 100: ...measurement using a specific AUFS on channel A Example While operating in single wavelength mode set an AUFS of 2 0000 on the second channel This provides 1 000 V AU on the channel B 2 V output The d...

Page 101: ...nnel appears 5 Enter the operating parameters for the second wavelength to be monitored including any timed and threshold events if desired For more information on operating the detector in single wav...

Page 102: ...minimum ratio and maximum ratio parameters on absorbance screen 5 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 a...

Page 103: ...econdary functions of the absorbance screen on page 3 18 3 In the Data out field press 5 MaxPlot A B 4 Press Enter to select the MaxPlot function 5 Return to the absorbance screen by pressing the HOME...

Page 104: ...the remote icon containing the letter E appears see the table on page 3 4 Timed events You can program up to 50 timed events to the nearest 0 01 minute As you enter timed events each new event is appe...

Page 105: ...he event appears 3 Enter the time for the event When you begin entering the time additional fields appear 6 Auto zero Does not apply Does not apply Yes 7 Lamp 1 Off 2 On Off No 8 Switch 1 1 High 2 Low...

Page 106: ...nt programmed 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 Tip ON A or ON B indicates the channel the event...

Page 107: ...both 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...

Page 108: ...er to display the threshold events choice list or press the number corresponding to the event you are programming see the table above 5 When the To field is active press Enter to display the options i...

Page 109: ...choice list by pressing METHOD Shift A B 2 Press 3 Retrieve a method The last method number stored or retrieved appears in the method number slot box 3 Enter the number of the method you want to retr...

Page 110: ...od the asterisk appears Method indicating that the method is 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 st...

Page 111: ...you can restore the previous conditions 4 Press 4 Store method and enter a storage location number To clear other stored methods repeat this step until you have cleared all desired methods 5 When you...

Page 112: ...e 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 Scanning using the cuvette on page 3 59 and Scanning usi...

Page 113: ...solution in comparison with the original scan done at a pace of 100 nm min Scan of anthracene at 100 nm min and 1000 nm min Tip The higher the number you enter in the Pace field the lower the resoluti...

Page 114: ...nd the same scan without tickmarks AUFS The sensitivity setting for scaling the charted spectrum Scan of erbium standard in a cuvette from 190 nm to 600 nm at a pace of 200 nm min with tickmarks every...

Page 115: ...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...

Page 116: ...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 di...

Page 117: ...scans of anthracene overlaid one at 100 nm min and one at 1000 nm min Tip The higher the number you enter in the Pace field the lower the resolution of the scan 6 Press Next to advance to the third Z...

Page 118: ...changes the value to OFF Refer to the figures on page 3 46 for examples of scans made with tickmarks on or off 10 Press Run to start the zero scan or press Next to return to the first zero scan parame...

Page 119: ...ctra 3 51 Zero and sample scan screen Sample scan Screen 1 of 3 Sample scan Screen 2 of 3 Sample scan Screen 3 of 3 Zero scan Screen 3 of 4 Zero scan Screen 4 of 4 Zero scan Screen 2 of 4 Zero scan Sc...

Page 120: ...The parameters for wavelength range AUFS Pace and Mark tickmark you entered for the corresponding zero scan appear 3 Press Next to advance to the second sample scan screen If desired you may change th...

Page 121: ...ical display 7 Press Scale Shift TRACE to change the scale zoom in on one part of the display You can alter the following four scaling parameters 1 minimum wavelength displayed 2 maximum wavelength di...

Page 122: ...t again to return to the sample scan display 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 Fo...

Page 123: ...to 300 nm 0 001 AU to 0 5 AU anthracene 230 to 270 nm Zoom of sample scan 230 nm to 270 nm 0 001 AU to 0 5 AU anthracene 250 nm Zoom of sample scan 300 nm to 400 nm 0 001 AU to 0 025 AU anthracene 330...

Page 124: ...ress 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 Press SCAN Shift Chart Mark to view the S...

Page 125: ...4 Press Enter The message Retrieving spectrum n appears After a brief wait the stored spectrum appears Once you retrieve a spectrum for review you can view it graphically and adjust the wavelength an...

Page 126: ...k the selected spectrum in real time both on the detector display and out the analog connector to the chart or A D device in a data collection system Once you retrieve a spectrum for playback the dete...

Page 127: ...site of both the contents of the cuvette and the flow cell you need to perform cuvette scans under identical flow cell conditions If you store spectra and acquire new spectra for subtraction you need...

Page 128: ...sample scans Before beginning a scan using the cuvette 1 Flush the flow cell with the eluent you are going to scan 2 To ensure a clear and clean cuvette wipe the clear portion of the cuvette with low...

Page 129: ...erted into the holder you can see liquid through the cuvette holder aperture that is the liquid completely covers the aperture Because the cuvette holder is angled use your thumb or forefinger to ensu...

Page 130: ...can using a manually filled flow cell Requirement Before you scan using a flow cell make sure there is no cuvette in the cuvette holder To run a spectrum using a flow cell 1 With a syringe fill the fl...

Page 131: ...amp 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 th...

Page 132: ...ate 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 programming it to turn on and off automatically for exa...

Page 133: ...the system for 10 minutes at 3 mL min 2 Replace the 100 water mobile phase with a solution of 90 methanol 10 water and flush the system for 10 minutes at 3 mL min Follow the recommended procedures fo...

Page 134: ...3 66 Preparing the Detector...

Page 135: ...e uncertain about how to perform the procedures call the Waters Technical Service Department to have a trained service representative perform the procedure Contents Topic Page Contacting Waters Techni...

Page 136: ...nce procedures in this chapter on your detector keep the following safety considerations in mind Spare parts Stock the recommended spare parts to minimize downtime Refer to Appendix C for a list of re...

Page 137: ...it 2 Gently remove the top of the cover and store it nearby The figure below illustrates the detector with the front left panel cover removed Detector with front left front panel cover removed Caution...

Page 138: ...ent 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 prob...

Page 139: ...re powering off Tip 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 3 Flush the detector with...

Page 140: ...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...

Page 141: ...stic tweezers Powder free finger cots or gloves Lint free swab Ethyl alcohol or methanol Nitrogen Save all parts after removal unless otherwise indicated Most of the removed parts are needed when you...

Page 142: ...4 8 Maintaining the Detector Unscrewing the flow cell assembly captive screws 5 Gently pull the assembly toward you Captive screws 3...

Page 143: ...Maintaining the flow cell 4 9 Removing the flow cell assembly 6 Once you remove the flow cell assembly from the detector chassis remove the cuvette holder from the flow cell assembly TP02813...

Page 144: ...se components Flow cell body Cuvette lens Split ring cuvette lens holder Cuvette lens screw Lens mount screw Exit window Exit window mount Caution To prevent contamination use powder free finger cots...

Page 145: ...o 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 cuvett...

Page 146: ...he entrance lens from inside the entrance lens screw 9 Using the plastic tweezers or the lint free swab tap and remove the clear plastic gasket The figure below shows an exploded view of all parts of...

Page 147: ...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 Using...

Page 148: ...ter 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...

Page 149: ...g from the main column connection and cap To replace the flow cell 1 Using a 1 4 inch flat blade screwdriver loosen the three captive screws on the flow cell assembly front plate see the figure on pag...

Page 150: ...10 This section describes the procedure for removing and replacing the detector deuterium lamp Lamp characteristics The intensity of the deuterium source lamp varies by wavelength as shown in the fig...

Page 151: ...al In this way the instrument s sensitivity to lamp energy is virtually eliminated as a major contributor to performance When operating the detector regularly invoke the lamp optimization software rou...

Page 152: ...r from the date of purchase whichever comes first Removing the lamp To remove the lamp 1 Extinguish the lamp using the keypad by pressing Shift Lamp and then pressing Shift Lamp again Tip Using the ke...

Page 153: ...mp to cool for a minimum of 30 minutes 4 Remove the front left panel cover as described in Proper operating procedures on page 4 3 5 Disconnect power to the lamp and disconnect the cord Lamp assembly...

Page 154: ...Pull the lamp assembly out of the lamp housing 8 Gently pull the lamp out Caution Lamp gas is under slight negative pressure To prevent shattering the glass use care when disposing of the lamp Cushion...

Page 155: ...lamp with a gas duster or lens tissue Warning To avoid the possibility of exposing your eyes to ultraviolet radiation never ignite a lamp while it is outside the instrument or if it is not properly s...

Page 156: ...g There is no additional alignment required 2 Gently push the lamp forward until it bottoms into position 3 Tighten the two captive screws 4 Reconnect the lamp power connector 5 When ready to resume o...

Page 157: ...age and its number of ignitions To record the new lamp serial number 1 Once the unit warms up press the DIAG key 2 Press 4 Lamp display keypad 3 Press 1 Change lamp Caution Always run the Change Lamp...

Page 158: ...month and move to the date field 7 Enter the number for the day of the month the lamp is installed and then press Enter to enter the date and move to the Year field 8 Enter the year last two digits on...

Page 159: ...es with the new lamp installed recalibrate the detector or recycle power to the detector after replacing the lamp Setting the lamp threshold You can set an alarm threshold for the lamp When the number...

Page 160: ...reen Replacing fuses The fuse holder is located on the rear panel of the detector see figure below Two fuses are shipped in place with the detector rated as indicated in Power requirements on page 2 5...

Page 161: ...en 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...

Page 162: ...3 Remove and discard the fuses 4 Make sure that the new fuses are properly rated for your requirements and then insert them into the holder and the holder into the power entry module gently pushing un...

Page 163: ...s to troubleshoot system problems Error messages Describe powerup calibration and other error messages and recommended actions for correcting the errors Diagnostics Describe user diagnostic tests for...

Page 164: ...methanol or water and the front left panel cover is attached securely The tables in this section are organized as follows Messages requiring you to perform corrective action These include messages enc...

Page 165: ...ngths 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...

Page 166: ...rm manual calibration procedure Peak not found Erbium n nm Erbium filter calibration range does not contain a local maximum 1 Remove cuvette 2 Be sure the front left panel door is closed 3 Flush flow...

Page 167: ...o cuvette in the cuvette holder and the empty cuvette holder is securely in place the flow cell is clean Calibration differs n nm At startup the unit performs a complete verification which includes re...

Page 168: ...ce Method not found Stored method data are invalid Recycle power to the detector This action removes the error Scan not found Stored scan data are invalid Recycle power to the detector This action rem...

Page 169: ...No response Unit sensors cannot identify any dark regions 1 Recycle power to the detector 2 Contact Waters Technical Service Filter initialization failure Shutter position Unit sensors cannot find th...

Page 170: ...vice Communication failure Reference A D A D communication test failed 1 Recycle power to the detector 2 Contact Waters Technical Service Communication failure Sample A D A D communication test failed...

Page 171: ...choices are indicated by Sticky diagnostic tests remain in effect until you disable them When a sticky diagnostic test is active the detector absorbance screen displays a wrench icon see the figure b...

Page 172: ...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 recycle power to the detector Detector...

Page 173: ...ubsidiary or your local Waters Technical Service Representative or call Waters corporate headquarters for assistance at 1 508 478 2000 U S Using the sample and reference energy diagnostic tests You us...

Page 174: ...the corresponding sample and reference energies appear 4 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 t...

Page 175: ...nels by pressing Cancel Shift 0 Auto Zero Offsets diagnostic display Setting fixed absorbance value From the Input Output choice list press 2 Fix absorbance to set a fixed absorbance value for channel...

Page 176: ...Voltage diagnostic display The voltage is driven on the selected analog channel A or B This is a sticky diagnostic To monitor contact closures and setting switches 1 From the Input Output choice list...

Page 177: ...ests press DIAG and then press 4 Using the Change Lamp diagnostic test To change the lamp 1 From the Lamp display keypad choice list press 1 Change lamp to access the Change Lamp diagnostic display Ch...

Page 178: ...espond when pressed contact your Waters service representative Rule You must press Enter twice to exit the keypad diagnostic display To use the Test Display diagnostic test 1 From the Lamp display key...

Page 179: ...ostic tests press DIAG and then press 5 Other diagnostics Other diagnostics choice list To generate test peaks 1 From the Other diagnostics choice list press 1 Generate test peaks to generate test pea...

Page 180: ...tical filter override to manually override the detector s automatic filter choice The Optical Filter Override diagnostic display appears Optical Filter Override diagnostic display 2 Press Enter to dis...

Page 181: ...l Service at 1 800 252 4752 U S and Canadian customers only Other customers call your local Waters subsidiary or your local Waters Technical Service Representative or call Waters corporate headquarter...

Page 182: ...r 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 troubleshootin...

Page 183: ...turn off the lamp or timed event within a method may be programmed to turn off the lamp No sample and reference energy Lamp burned out Attempt to reignite using the Lamp key Replace the lamp Lamp off...

Page 184: ...ve cuvette from cuvette holder 2 Be sure the flow cell mobile phase does not absorb above 250 nm 3 Ensure there are no air bubbles in the flow cell 4 Recalibrate the detector 5 If problems persist con...

Page 185: ...des 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 t...

Page 186: ...ks 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 spec...

Page 187: ...around 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 tetrahydro...

Page 188: ...al surfaces of the mass spectrometer when the instrument 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 v...

Page 189: ...When operating this device follow standard quality control procedures and the equipment guidelines in this section Warning Waters instruments can be used to analyze or process potentially hazardous s...

Page 190: ...n 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...

Page 191: ...e 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 n...

Page 192: ...no rigonfiamento nei tubi non metallici riducendo notevolmente la resistenza alla rottura dei tubi stessi Advertencia se recomienda precauci n cuando se trabaje con tubos de pol mero sometidos a presi...

Page 193: ...Warnings that apply to all Waters instruments A 9...

Page 194: ...isque d tre d fectueuses Vorsicht Der Benutzer wird darauf aufmerksam gemacht dass bei unsachgem er Verwenddung des Ger tes unter Umst nden nicht ordnungsgem funktionieren Attenzione l utente deve ess...

Page 195: ...de la m me puissance afin d viter tout risque d incendie Vorsicht Zum Schutz gegen Feuergefahr die Sicherungen nur mit Sicherungen des gleichen Typs und Nennwertes ersetzen Attenzione per una buona p...

Page 196: ...prise avant d effectuer la maintenance de l instrument Vorsicht Zur Vermeidung von Stromschl gen sollte das Ger t vor der Wartung vom Netz getrennt werden Attenzione per evitare il rischio di scossa e...

Page 197: ...ls These can appear in instrument user manuals and on the instrument s front or rear panels Electrical power on Electrical power off Standby Direct current Alternating current Protective conductor ter...

Page 198: ...visories Handling symbols These handling symbols and their associated text can appear on labels affixed to the outer packaging of Waters instrument and component shipments Keep upright Keep dry Fragil...

Page 199: ...ions 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...

Page 200: ...nitrile analytical cell Dual channel noise dry 12 AU at 230 nm 280 nm with 2 second filter at 1 Hz data rate dry cell Dual channel noise wet 15 AU at 230 nm 280 nm with 2 second filter at 1 Hz data ra...

Page 201: ...r on demand Flow cell TaperSlit flow cell design Nitrogen purge Purge fitting present on optics bench Path length 10 mm standard analytical Cell volume 10 L standard analytical Pressure limit 1000 psi...

Page 202: ...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...

Page 203: ...sure Rating In Out psi bar Analytical Cell 10 10 0 009 0 009 1000 70 Semi 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 Hig...

Page 204: ...B 6 Detector Specifications...

Page 205: ...000186 Analytical TaperSlit Flow Cell Assembly WAS081140 General Parts Waters Startup Kit 2489 Detector 200000227 Cuvette Holder Assembly WAS081333 Dual Detector Wavelength Accuracy Cuvette Kit WAT047...

Page 206: ...High Pressure 3000 psi Cell for LCMS WAT081321 Autopurification Cell 289000614 Optional Variable Prep Flow Cells Variable Prep Flow Cell 700000923 Variable Prep Flow Cell Quartz Rods and Seals Kit 700...

Page 207: ...nt miscibility D 3 Buffered solvents D 6 Head height D 6 Solvent viscosity D 6 Mobile phase solvent degassing D 6 Solvent degassing methods D 8 Wavelength selection D 9 Warning To avoid chemical hazar...

Page 208: ...s generally maintain 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 go...

Page 209: ...nol temperature affects solvent miscibility If you are running a high temperature application consider the effect of the higher temperature on solvent solubility buffers dissolved in water can precipi...

Page 210: ...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...

Page 211: ...icity scale These solvents receive a dual 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 e...

Page 212: ...Generally viscosity is not important when you are operating with a single solvent or under low pressure However when you are running a gradient the viscosity changes that occur as the solvents are mix...

Page 213: ...olecular attractive forces similar to those in the gas like dissolves like Effects of temperature Temperature affects the solubility of gases If the heat of solution is exothermic the solubility of th...

Page 214: ...atmospheric gases Sparging can change 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 solven...

Page 215: ...mber 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...

Page 216: ...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 k...

Page 217: ...Phase UV Cutoff nm Mobile Phase UV Cutoff nm Acetic acid 1 230 Sodium chloride 1 M 207 Ammonium acetate 10 mM 205 Sodium citrate 10 mM 225 Ammonium bicarbonate 10 mM 190 Sodium dodecyl sulfate 190 BR...

Page 218: ...cm Ether O 185 1000 Thioether S 194 4600 215 1600 Amine NH2 195 2800 Thiol SH 195 1400 Disulfide S S 194 5500 255 400 Bromide Br 208 300 Iodide I 260 400 Nitrile C N 160 Acetylide C C 175 180 6000 Sul...

Page 219: ...llene C C 2 acyclic 210 230 21 000 Allene C C 3 260 35 000 Allene C C 4 300 52 000 Allene C C 5 330 118 000 Allene C C 2 alicyclic 230 260 3000 8000 Ethylenic Acetylenic C C C C 219 6 500 Ethylenic Am...

Page 220: ...D 14 Solvent Considerations...

Page 221: ...3 4 navigating from 3 12 secondary functions 3 13 accessing secondary functions 3 12 activating a pulse or rectangular wave 3 24 active method 3 41 additional functions in dual wavelength mode 3 32 ad...

Page 222: ...ace 3 9 time constant 3 12 wavelength from dual to single 3 8 3 33 wavelength from single to dual 3 8 3 33 channel changing 3 9 I and II outputs 2 10 selector 3 4 chart mark configuring event inputs 3...

Page 223: ...xt sensitive help 3 26 contrast adjusting 3 25 changing 3 11 function 3 25 Contrast key 3 11 controlling from Waters data systems 2 14 cover removing 4 3 current method conditions 3 13 3 35 3 43 cuvet...

Page 224: ...erride 5 11 5 18 procedure 5 9 5 18 reset 5 10 sample and reference energy 5 10 5 11 service 5 11 5 19 setting a fixed absorbance value 5 13 setting a fixed voltage output 5 14 startup 3 2 sticky 3 5...

Page 225: ...ilure of startup diagnostic tests 3 3 features design 1 2 MaxPlot function 1 15 RatioPlot function 1 15 spectral 1 10 filter time constant changing 3 12 function 3 16 parameter 3 19 filters filter set...

Page 226: ...trace 3 20 3 22 zoom 3 20 fuse holder 4 26 replacing 4 26 4 28 G gas solubility D 7 generate test peak diagnostic test 5 11 generating auto zero on inject from the 2695 2 18 chart mark from the 2695 2...

Page 227: ...on 3 8 DIAG key 3 9 diagnostic test 5 11 5 16 Enter key 3 11 functions 3 6 3 8 help key 3 8 3 26 HOME key 3 8 key 3 8 3 31 3 33 Lamp key 3 10 lamp display and keypad diagnostic tests 5 11 5 15 Lock ke...

Page 228: ...ometer shock hazard A 4 maximum ratio field 3 35 function 3 16 parameter 3 20 MaxPlot function charting 3 15 features 1 15 obtaining 3 35 method active 3 41 choice list 3 9 current conditions 3 13 ini...

Page 229: ...36 Ethernet connections 2 14 2 15 output connections 2 10 signals 2 13 overriding the optical filter 5 18 P pace 3 44 parameters absorbance threshold timed event 3 37 analog out dual wavelength 3 19 A...

Page 230: ...fuses 4 26 illustration 2 10 signal connections 2 11 reassembling the flow cell 4 6 4 15 rebuild kit flow cell 4 11 rebuilding the flow cell 4 13 4 14 recalling the absorbance screen using the HOME k...

Page 231: ...key 3 8 3 47 Scanning subtracting 3 57 scanning Anthracene 3 45 3 55 AUFS 3 46 cuvette 3 44 3 59 3 62 Erbium 3 46 flow cell 3 44 3 62 initiating 3 8 new spectra 3 48 3 54 pace 3 44 reference energy 3...

Page 232: ...ion 3 28 degasser 3 26 degassing 4 4 filter 4 4 general considerations D 2 D 3 guidelines D 2 miscibility D 3 D 5 reservoirs D 6 UV cutoff D 9 D 11 viscosity considerations D 6 spare parts 4 2 C 1 spa...

Page 233: ...aks generating 5 17 The 4 15 theory of operation 1 1 1 15 threshold events clearing 3 43 programming 3 39 3 40 tickmarks generating 3 46 time constant changing 3 12 function 3 16 timed event parameter...

Page 234: ...ark connections 2 31 configuring the detector lamp signal 2 29 connecting to 2 29 2 33 Inject Start connections 2 32 Waters 717plus Autosampler auto zero connections 2 34 connecting to 2 33 2 35 Injec...

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