Waters 2475 Operator'S Manual Download Page 15

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1-1

Theory of Operation

This chapter explains the theory and technology supporting the
operation of the Waters

2475 Multi

Fluorescence Detector and

describes the instrument’s features.

Contents:

Topic

Page

Fluorescence theory

1-2

Fluorescence detection

1-3

Measuring fluorescence

1-4

Detector description

1-7

Principles of operation

Lamp energy and performance

1-16

Auto-optimize gain

1-17

Startup diagnostic tests

1-21

Mobile-phase solvent degassing

1-21

Wavelength selection

1-22

Summary of Contents for 2475

Page 2: ...t 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...

Page 3: ...disposal of any Waters product You can reach us via the Internet telephone or conventional mail Safety considerations Some reagents and samples used with Waters instruments and devices can pose chemi...

Page 4: ...ted in this section Applicable symbols Audience and purpose This guide is intended for personnel who install operate and maintain the 2475 Multi Fluorescence detector Symbol Definition Manufacturer Au...

Page 5: ...ne 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 with...

Page 6: ...vi 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...

Page 7: ...trument v Calibrating v Quality control v ISM classification v ISM Classification ISM Group 1 Class B v EC authorized representative vi 1 Theory of Operation 1 1 Fluorescence theory 1 2 Fluorescence d...

Page 8: ...12 Electronics 1 13 Wavelength verification and test 1 14 Operational modes 1 14 Single channel mode 1 14 Multichannel mode 1 15 Spectrum scanning 1 16 Lamp energy and performance 1 16 Auto optimize g...

Page 9: ...20 Connecting other devices 2 23 Required materials 2 23 Connecting cables 2 23 Connecting a data system using a Bus SAT IN module 2 24 Connecting a 746 data module 2 27 Connecting a chart recorder 2...

Page 10: ...ion mode via RS 232 3 29 Remote control operation via Ethernet connection using 2475 instrument control software 3 34 Verifying the detector 3 34 Manual wavelength calibration 3 35 Normalizing emissio...

Page 11: ...68 Manually lighting the lamp 3 69 Using a timed event method to program the lamp 3 70 Shutting down the detector 3 70 4 Maintenance Procedures 4 1 Contacting Waters technical service 4 2 Maintenance...

Page 12: ...iagnostic tests and settings 5 12 Change lamp function 5 15 Testing the keypad 5 16 Testing the display 5 17 Other diagnostic tests and settings 5 17 Generating test peaks 5 17 Overriding the optical...

Page 13: ...ation checklist C 2 Water C 2 Buffers C 3 Tetrahydrofuran THF C 3 Solvent miscibility C 3 How to use miscibility numbers C 5 Buffered solvents C 6 Head height C 6 Solvent viscosity C 6 Mobile phase so...

Page 14: ...xiv Table of Contents...

Page 15: ...strument s features Contents Topic Page Fluorescence theory 1 2 Fluorescence detection 1 3 Measuring fluorescence 1 4 Detector description 1 7 Principles of operation 1 9 Operational modes 1 14 Spectr...

Page 16: ...erfere with a compound s ability to fluoresce diminishing analytical performance pH changes The loss or gain of protons and their accompanying increase or decrease of charge affects an analyte s elect...

Page 17: ...ight sources Xenon lamps are the preferred source for general purpose fluorescence detectors Excitation wavelength selection The excitation wavelength of choice requires some source light filtering In...

Page 18: ...nt of stray light that can affect the measurement and it maximizes the fluorescence signal The sample compartment is arranged so that the fluorescence energy is collected at an angle perpendicular to...

Page 19: ...he 2475 detector reports intensity using normalized units to compensate for variability between individual detectors and offset any age related decrease in lamp intensity When using normalized units c...

Page 20: ...iables such as lamp intensity optics efficiency and gain directly influence the fluorescence emission signal strength As a result energy units are less reliable Nonetheless when you must calculate ene...

Page 21: ...Interscience New York 1972 Detector description The 2475 Multi Fluorescence Detector is a multichannel tunable fluorescence detector designed for high performance liquid chromatography HPLC applicati...

Page 22: ...ols to optimize functionality and performance Data communications and control By an Empower system or Millennium 32 chromatography workstation version 3 2 or 4 0 Backward compatibility Operates as a 4...

Page 23: ...verification and test Flow cell Electronics Detector optics The optics are based on a pair of tunable monochromators and include the following parts Xenon arc lamp Two ellipsoidal mirrors and one par...

Page 24: ...ly light path The detector demonstrates superior performance by employing several unique design elements Its novel flow cell design minimizes stray background light Xenon lamp Grating Parabolic mirror...

Page 25: ...nochromator to select the appropriate excitation wavelengths defined by its geometry The grating can rotate quickly responding to multiple excitation wavelengths and or scanning Emission monochromator...

Page 26: ...sensitivity After calibrating the PMT you must choose a gain setting for the photomultiplier tube prior to a chromatographic injection Saturation which occurs when the sample concentration is high or...

Page 27: ...erformed simultaneously This component ensures the best rejection of common mode noise in the two beams leading to a quiet baseline Personality board Receives inputs from the preamplifier board and ex...

Page 28: ...nt mobile phase You can initiate a manual wavelength calibration anytime to replace the previous calibration data with new data Tip The combined wavelength accuracy specification of the detector is 3...

Page 29: ...fy a higher sampling rate in the instrument method If the value is greater than 50 you should specify a lower sampling rate in the instrument method Set the sampling rate to the lowest value required...

Page 30: ...by performing the following types of scan on the flow cell Zero scan Characterizes the baseline spectrum of a solvent Excitation sample scan Subtracts the zero scan so the displayed or charted result...

Page 31: ...ed the detector to operate as independently of lamp output as possible After the unit has verified the calibration of the monochromator the instrument evaluates the energy levels in a number of charac...

Page 32: ...alog outputs during data collection it displays a minimum EUFS value that applies to the entire chromatogram Like the ideal gain value the EUFS value assumes a 2 margin to account for any variations i...

Page 33: ...small peak that is best detected at a gain of 1000 The next change at 2 0 minutes is the required wavelength pair change Initial gain setting or conditions are not critical The only requirement for th...

Page 34: ...ple energy units changing the gain does affect the magnitude of the output signal Ensuring gain optimization for each peak of interest Refer to the figure Gain optimized chromatogram on page 1 19 If y...

Page 35: ...any test fails The startup diagnostic tests are as follows Central processing unit CPU test Serial communication interface SCI test Electrically erasable programmable read only memory EEPROM test RAM...

Page 36: ...solvent absorbs some of the available excitation light intensity which in turn reduces the fluorescence emission response for the sample For a complete list of UV cutoff ranges for common solvents and...

Page 37: ...Up the Detector Contents Topic Page Before you begin 2 2 Installing the detector 2 3 Plumbing the detector 2 3 Making signal connections 2 5 Connecting other devices 2 23 Connecting to the electricit...

Page 38: ...and your local Waters representative Customers in the USA and Canada should report damage and discrepancies to Waters Technical Service 800 252 4752 Others should phone their local Waters subsidiary...

Page 39: ...irmly and then tighten the compression screw Warning To avoid injury Waters recommends that two people lift the 2475 detector Warning Risk of fire To avoid overheating and to provide clearance for cab...

Page 40: ...nections Assembling fittings Slide the compression screw over the tubing end Follow it with the ferrule mounted so that its taper faces the end of the tubing Compression screw Tube Ferrule Tubing end...

Page 41: ...ht Tip To ensure accurate verification during installation be sure to pump fresh degassed and filtered 100 water through the flow cell before powering it Making signal connections See also Ethernet In...

Page 42: ...summarizes the signal connections needed to connect the 2475 detector to other HPLC system components Component connector types Connector type Component Ethernet connection Used to connect to a Water...

Page 43: ...accompany the instrument control software for details Single Waters instrument connection In a single Waters instrument system configuration the connection hardware requires only one CAT 5 shielded Et...

Page 44: ...u must install the Waters instrument control software in the acquisition computer so that the computer can control the Waters instrument Consult the software installation instructions that accompany t...

Page 45: ...mmarizes the inject start connections for different system configurations 2475 detector inject start connections Inject start output source Inject start input connection on the 2475 detector connector...

Page 46: ...led according to the EUFS emission energy units full scale setting for each channel The detector allows the EUFS to be set individually for the output on each channel Volts per EU are calculated for t...

Page 47: ...cribes each signal available on the I O connectors See Appendix B for the signal s electrical specifications I O signals for the detector Signal Description Inject Start 1 TTL contact closure Configur...

Page 48: ...og output channels Detector Out 1 and Detector Out 2 Auto Zero 1 Configurable input to Auto Zero both channels Detector Out 1 and Detector Out 2 Detector Out 1 2 1 V full scale analog output signal of...

Page 49: ...ads of the signal cable to the connector 2 Slide the clamp with the bend facing down into the protective shield 3 Insert the clamp and shield with the bend facing down into the connection cover and lo...

Page 50: ...second connection cover over the first cover and snap it into place Connecting an Alliance separations module You can connect an Alliance separations module to the detector to perform the following fu...

Page 51: ...r auto zero on injection Connections for generating an auto zero on inject Alliance separations module B inputs and outputs 2475 detector A inputs Pin 1 Inject Start Pin 9 Auto Zero Pin 2 Inject Start...

Page 52: ...n injection Connections for generating a chart mark on inject Alliance separations module B inputs and outputs 2475 detector A inputs Pin 1 Inject Start Pin 6 Chart Mark Pin 2 Inject Start Pin 7 Chart...

Page 53: ...ging the default lamp configuration parameter from Ignore to High or Low see page 3 25 Connections for starting a method Alliance separations module B inputs and outputs 2475 detector A inputs Pin 1 I...

Page 54: ...tions port in an Empower system or Millennium 32 chromatography Connections for turning the detector lamp on or off Alliance separations module A Outputs 2475 detector A Inputs Pin 1 Switch 1 Pin 4 La...

Page 55: ...onnecting the detector to a Waters data system all detector parameters not configurable by the data system in use defer to local control Caution To avoid possible damage to components shut down all in...

Page 56: ...ctions for starting a method on injection on page 2 17 Connecting Ethernet devices The detector can be controlled using Ethernet communications when Empower software build 2154 or build 1154 with Inst...

Page 57: ...ge 2 22 You must install the Waters instrument control software in the acquisition computer before the computer can control the Waters instrument See the software installation instructions that accomp...

Page 58: ...or different system configurations Tip For pin out connections to the 2475 detector see the figure I O signal inputs and outputs on page 2 11 2475 detector inject start connections Inject start output...

Page 59: ...you need the following tools Small flat blade screwdriver startup kit Electrical insulation stripping tool Connecting cables To connect the cables from other HPLC system devices to the A and B termina...

Page 60: ...nel To connect an Empower system or Millennium32 chromatography workstation to the detector 1 Connect the Bus SAT IN module to the busLAC E or LAC E32 card in an Empower system or Millennium 32 comput...

Page 61: ...ting the Bus SAT IN module channel 2 to the detector on page 2 26 and the figure I O signal inputs and outputs on page 2 11 Connect the white wire to pin 4 on B Detector Out 2 1 V Connect the black wi...

Page 62: ...SAT IN module 2475 detector B inputs and outputs 1 2 3 4 5 6 7 8 9 10 Detector out 1 Detector out 1 Ground Detector out 2 Detector out 2 Switch 1 Switch 1 Ground Switch 2 Switch 2 Bus SAT IN module 24...

Page 63: ...module use the cable provided in the detector s startup kit to make the connections shown in the following table and figure Tip To minimize the chance of creating a ground loop that can adversely affe...

Page 64: ...and figure Tip 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 Detector inputs and chart r...

Page 65: ...generate a chart mark A chart mark signal is generated at the chart mark inputs on the analog connector Connecting a 600 series pump To connect a 600 series pump 1 Place the detector on a level surfa...

Page 66: ...ontroller to the detector shown in the following table and figure Lamp on off connections for the 600 series pump Detector inputs and 600 series pump terminal connections 600 series pump terminal 2475...

Page 67: ...t mark connections To make chart mark connections 1 Make the connections shown in the following table and figure using a signal cable Auto zero connections for the 600 series pump 600 series pump term...

Page 68: ...nitiate data acquisition To make inject start connections 1 Make the connections shown in the following table and figure with a signal cable Chart mark connections for the 600 series pump 600 series p...

Page 69: ...zero at the start of an injection Inject start connections for the 600 series pump 600 series pump terminal 2475 detector A inputs S1 S2 S3 S4 1 or inject 1 You may also connect the pump s Inject ter...

Page 70: ...autosampler Auto zero connections for the 717plus autosampler Auto zero connections for the 717plus autosampler 717plus autosampler terminal 2475 detector A inputs Inject Start any one of many paired...

Page 71: ...art terminals on the autosampler Inject start connections for the 717plus Autosampler Inject start connections for the 717plus Autosampler 717plus Autosampler terminal 2475 detector A inputs Inject St...

Page 72: ...bility 1 Place the Off On switch in the Off position 2 Connect the female end of the power cord to the receptacle on the rear panel of the detector 3 Connect the male end of the power cord to a suitab...

Page 73: ...in the Empower Online Help to set parameters for controlling the detector As part of a Millennium 32 chromatography workstation You can configure the detector for use with a Millennium 32 chromatograp...

Page 74: ...ges 1 Initializing grating 2 Initializing system 3 Lighting lamp 4 Warmup time left counts down from five minutes Warning Always observe Good Laboratory Practices when you use this equipment and when...

Page 75: ...p failure If one or more of the internal startup checks fails the detector beeps and displays an error message For serious errors the detector displays the word Error in brackets Error in place of the...

Page 76: ...V light from reaching the detector s optics bench 2475 detector idle mode screen Using the operator interface Using the display The detector s operator interface includes a 128 64 bitmap graphic displ...

Page 77: ...play the icons or fields shown on page 3 4 and described in the following table For a list of ranges and defaults for the function icons and fields in the following table see the table titled Primary...

Page 78: ...Channel trace When you press TRACE displays the fluorescence intensity also known as emission for the channel indicated A or B Numerical field 0 00 Fluorescence in emission units or sample energy unit...

Page 79: ...erating in triple channel mode Icon only appears if instrument is under control of an external data system remote mode only Quad wavelength Indicates the detector is operating in quadruple channel mod...

Page 80: ...current method number or an asterisk which indicates current conditions are not stored as a method RS 232 control Indicates that the 2475 detector is controlled by a data system and displays a remote...

Page 81: ...ggles between channels A and B Direct access to specific screens HOME METHOD CONFIGURE DIAG Diagnostics TRACE and SCAN Primary functions Chart Mark Auto Zero and Run Stop Primary function keys take ef...

Page 82: ...entry on a list or menu as follows For numerical entries 1 through 9 on lists or menus enter the number corresponding to a desired item and then press Enter For the number 10 select 0 and then press...

Page 83: ...d description Key Description Unshifted After pressing shift HOME Displays the home screen which displays icons excitation and emission wavelengths EUFS and Gain fields Displays context sensitive Help...

Page 84: ...ps freezes the run clock and initiates scans The elapsed time appears near the lower right hand side of the home screen The shutter opens when you press Run while the idle mode function is enabled Res...

Page 85: ...s for creating and clearing timed and threshold events and storing retrieving and resetting methods DIAG Displays the list of diagnostic routines CONFIGURE Displays the first Configuration screen TRAC...

Page 86: ...of the lamp is indicated by an icon on the home screen 2 See 0 9 above Lock When you are viewing the home screen enables or disables the keypad lock feature Use the lock to prevent inadvertent changes...

Page 87: ...value Sets the value to a unique word for some fields For example in the voltage offset diagnostic you can enter either a numeric offset value or press CE to change it to OFF Clear Field Blanks the c...

Page 88: ...e number corresponding to a choice you can press that number without pressing Enter first The and keys do not incrementally increase or decrease numerical field entries To change field entries use the...

Page 89: ...figure Secondary functions of the home screen on page 3 17 Secondary functions of the home screen Fluorescence home screen Press Next filter type analog out and time constant available for both and P...

Page 90: ...he shutter by pressing Reset or waiting until the run ends To avoid the automatic opening and closing of the shutter you can disable the idle mode by checking the Selection box in the Configuration sc...

Page 91: ...setting must have a linear relationship with the actual fluorescence signal Filter type Defines your choice of noise filter Hamming filter is the default Data units Defines data units Emission The st...

Page 92: ...nnel regardless of which channel is larger Volts out Larger fluorescence A or B 1 V EUFS of selected channel Difference Plot A B and B A Charts the difference in fluorescences at two different wavelen...

Page 93: ...eline maintains the previous baseline level when the gain or wavelength change is made To baseline is the default setting Enable keypad event in chart mark Selected by default this parameter results i...

Page 94: ...nce energy A Output off Emission A Time constant 2 of 4 Numeric sec Hamming 0 1 to 5 0 Hamming 1 to 50 RC 0 1 to 99 RC 1 to 99 0 to disable filtering 1 5 Data units 3 of 4 Choice None Emission Energy...

Page 95: ...tion of the trace Displaying the scale parameters To display the scale parameters 1 Press Scale 2 Press Next to display T2 starting time The default is 0 3 Press Next again to display EU1 starting or...

Page 96: ...tion wavelength set to 240 nm and emission to 355 nm Scaled trace of continuous injections with T1 changed to 60 The following figure shows a 4 minute scaled trace or zoom of the 60 minutes of continu...

Page 97: ...s Disabling shutter idle mode You can disable the shutter idle mode in the first Configuration screen When selected the shutter does not close to protect the detector s optics after the completion of...

Page 98: ...You can configure the auto zero input to auto zero fluorescence readings on channel A and or channel B To determine the response of the channel enable the auto zero function see the table titled Prima...

Page 99: ...is programmed to initiate a rectangular wave as a timed or threshold event then this field specifies the period of the signal the width of one pulse period in a rectangular wave or pulse train range...

Page 100: ...ive Help When you select Shift HOME from a point in the program associated with a Help screen the screen appears If Online Help is not available selecting effects no response Press Enter to return to...

Page 101: ...3 29 Standalone operation When using the detector as a standalone instrument you can store up to 10 methods each containing up to 48 timed and 2 threshold events see page 3 43 An asterisk in the metho...

Page 102: ...ilable Tip The same parameters apply to a 474 detector Method parameters The initial conditions for the method are specified on the General tab of the fluorescence method editor in Empower or Millenni...

Page 103: ...t a value close to the actual value in the method for sample detail archival purposes in Empower or Millennium32 software Filter Type Either a digital Hamming or RC filter The digital filter tends to...

Page 104: ...to wait for the lamp to warm again Tip It is best to shut the lamp off only when all runs are completed You should program the lamp to turn off or turn off the lamp manually only when you do not expe...

Page 105: ...wavelength or gain is changed Polarity Defines the polarity of the analog output only Operation details Inject Start The detector is configured with a switch closure signal input to the chart mark ter...

Page 106: ...are Remote control operation via Ethernet connection using 2475 instrument control software This mode of operation uses the Ethernet connection and appears in the Empower software Configuration window...

Page 107: ...gth 1 Select Calibrate Shift 3 from the keypad Wavelength calibration message 2 Ensure the flow cell is prepared and then press Enter The detector cycles through the calibration procedure and momentar...

Page 108: ...with filtered degassed and sparged HPLC grade water Then pump the mobile phase provided you encounter no miscibility problems at 1 mL min for 15 minutes minimum Tip Normalize emission units weekly To...

Page 109: ...operation the default operating mode To specify single channel mode when the detector is in multichannel mode 1 From the home screen select l Shift Auto Zero Result The detector displays an appropria...

Page 110: ...the detector with expanded chart out selections in multichannel mode which offers the following functions Emission A and B Sample energy A and B Reference energy A and B MaxPlot Difference A B or B A...

Page 111: ...not set the gain setting for each channel individually Obtaining a MaxPlot or difference plot You can obtain a MaxPlot or difference plot by monitoring fluorescence at two selected wavelengths while p...

Page 112: ...s the pre amplifier and a value of 9999 9 appears in the Emission Energy units field Gain set too high fluorescence pinned to 9999 9 EU Gain set too high alarm message The value of 9999 9 EU in the Em...

Page 113: ...am This can be done by entering a method via the keypad or by retrieving a previously created method from a stored memory location If you are using Empower or Millennium 32 software you must enter the...

Page 114: ...at proper times relative to the peaks Select Make Injection if you are running under Empower or Millennium32 control or start the injection through other devices such as an Alliance 2695 Separations M...

Page 115: ...on scale Gain and EUFS optimally set Programming methods and events Storing methods You can store and retrieve up to ten methods The detector designates stored methods as the numerals 1 through 10 If...

Page 116: ...down are restored However any timed events or thresholds associated with the method are deactivated when power is restored Thus on startup an asterisk always appears inside the method icon on the home...

Page 117: ...vity EUFS 1 to 100 000 Yes 6 Chart mark 10 of full scale Does not apply Does not apply Yes 7 Polarity 1 2 Yes 8 Auto Zero Does not apply Does not apply Yes 9 Lamp 1 Off 2 On Off No 10 Switch 1 1 High...

Page 118: ...ifying the time of the event appears 3 Specify the time for the event Tip When you begin additional fields appear Timed events screen 4 Press Enter To advance to the Set field Events list press 12 Thr...

Page 119: ...hen the time field is active to change it to OFF 9 Press HOME to return to the home screen then press Run Stop to start the method 10 Select Reset Shift Run Stop to reset the run clock to 0 Tip If the...

Page 120: ...vents An active field EU for entering the threshold appears When you begin to enter a number in the EU field additional fields appear Tip The threshold event allows you to specify a threshold value EU...

Page 121: ...to the threshold parameter you are programming 6 Press A B to set the threshold on the other channel and repeat the procedure Storing a method A method consists of all programmable parameters shown on...

Page 122: ...ot box 3 Specify the number of the method you wish to retrieve and press Enter A brief message Retrieving method n appears and then the method number you specified appears within the method number ico...

Page 123: ...her operating parameters x e UFS etc are set to defaults If you select Cancel Shift 0 the Method list appears Tip To prevent loss of the current conditions before you clear the method store them in a...

Page 124: ...solvent to provide the actual spectrum of the sample The detector can measure the spectrum of a sample using the flow cell see page 3 64 for scanning procedures Before you begin Before you run a spect...

Page 125: ...000 nm min Tick marks Generates tick marks at a specified wavelength interval which helps to interpret charted data The figure Scan of water without tick marks on page 3 54 shows a scan of water stand...

Page 126: ...e detector s scan function to run a new zero or sample scan store review subtract obtain scan information and replay When you select a sample or zero scan the detector displays three additional screen...

Page 127: ...tor displays one additional screen labeled 2 of 2 Note that you cannot change the starting parameter value ending wavelength parameter value or the pace parameter value Zero scan screen 1 of 4 Sample...

Page 128: ...the subsequent sample scans you perform The sample scan uses the starting and ending wavelength values and the pace value of the most recent zero scan Only when these parameter values are identical fo...

Page 129: ...eens 4 On the first New scan screen specify the type of scan For a sample scan press 1 or press Enter to display the list For a zero scan press 2 or press Enter to display the list The detector displa...

Page 130: ...ts Range or default Type 1 n a Sample scan 1 Zero scan 2 Default 1 range 2 nm Range 200 to 900 nm Default 200 or 210 nm Pace 3 nm min Range 30 to 1000 nm min Default 100 nm min EUFS 3 EU Range 1 to 10...

Page 131: ...alue and then press Enter 7 Specify a value in the Pace field for the rate at which the detector scans the specified wavelength range Tip The default is 100 nm min and the allowable range is from 30 t...

Page 132: ...and solvent conditions run the sample scan for the corresponding zero scan within 15 minutes of running the zero scan 1 Set up and run the zero or reference scan see page 3 59 2 Return to the first N...

Page 133: ...a sample scan the detector indicates that the zero scan is being subtracted from the sample scan in progress Subtracting the zero scan progress bar After the scan is complete the detector displays the...

Page 134: ...wavelength displayed EU1 Minimum fluorescence displayed The default is auto EU2 Maximum fluorescence displayed The default is auto 8 Press Next to advance through the four scaling parameters The follo...

Page 135: ...age 3 64 shows a series of scans of anthracene dissolved in acetonitrile The zero scan is not displayed For the scaling parameters EU1 and EU2 the default is auto You can change the EU parameter based...

Page 136: ...ell with the analyte 4 Run a sample scan keeping the pressure below 145 psi to avoid overpressuring the flow cell 5 Use the functions storage review subtract and review and replay to compare scanned d...

Page 137: ...pectrum being subtracted You can replay the current spectrum or a stored one in real time using the real time replay function on the scan list The detector plays the selected spectrum in real time bot...

Page 138: ...for the selected spectrum other The wavelength setting of the stationary monochromator Pace Displays the pace for the selected spectrum Gain The gain setting for the PMT it can sometimes be necessary...

Page 139: ...current spectrum and after a brief wait displays the difference spectrum You can store the results in one of the five storage slots Replaying a spectrum To replay a spectrum 1 Select SCAN Shift Chart...

Page 140: ...mp manually When the lamp is unlit the home screen displays the words Lamp off and an X is superimposed on the lamp icon Select the Lamp key Shift 1 to extinguish or light the lamp manually and to dis...

Page 141: ...ght the lamp 1 Select Lamp Shift 1 The lamp control screen appears with 0 hours and 00 minutes in the Lamp has been on field 2 Select Lamp Shift 1 again to light the lamp Tip When the lamp is lit the...

Page 142: ...ore information on programming the lamp through the external contact closure Shutting down the detector Before you shut down the detector you must remove any buffered mobile phase present in the fluid...

Page 143: ...ic Page Contacting Waters technical service 4 2 Maintenance considerations 4 2 Routine maintenance 4 3 Inspecting cleaning and replacing the flow cell 4 5 Replacing the lamp 4 8 Replacing the fuses 4...

Page 144: ...he symptom Instrument serial number Solvent s Method parameters sensitivity and wavelength Type and serial number of column s Sample type Empower software version and serial number For complete inform...

Page 145: ...is shut down Tip Flushing prevents the following problems Plugging of the solvent lines and flow cell Damage to various components Microbial growth Warning Avoid electric shock Do not open the detect...

Page 146: ...raw the top of the cover gently and store the cover nearby 2475 detector with the front left hand panel cover removed Caution Because no user serviceable parts are housed inside the detector never rem...

Page 147: ...Discontinue the flow of mobile phase 2 Remove the column 3 Attach tubing from the detector to the injector outlet from which the column has been removed 4 Flush the mobile phase from the detector wit...

Page 148: ...Flush and dry the flow cell see page 4 5 and then disconnect and cap the inlet and outlet LC tubing 3 Remove the front left hand panel cover 4 Use a 1 4 inch flat blade screwdriver to loosen the three...

Page 149: ...low cell installed Replace the flow cell when it becomes damaged Before you begin 1 Unpack and inspect the new flow cell 2 Shut down the detector 3 Remove the front left hand panel cover 4 Disconnect...

Page 150: ...stic tests for 2000 hours or 1 year from the date of purchase whichever comes first Tip Always perform the procedure on page 3 34 each time you change the lamp When to replace the lamp Replace the lam...

Page 151: ...extremely hot during lamp operation To prevent burn injuries allow the lamp to cool for 60 minutes before removing it keep the lamp in the housing when handling the lamp Warning To avoid eye injury f...

Page 152: ...4 10 Maintenance Procedures b Pinch the locking mechanism on the bottom connector before pulling it out Unplugging the lamp assembly...

Page 153: ...mbly Warning To avoid injury always keep the lamp facing away from you when removing it Lamp gas is under positive pressure To prevent shattering the glass use care when disposing of the lamp Waters s...

Page 154: ...place 5 Close and secure the lamp access door 6 Connect the power cord and start the detector Allow at least 60 minutes for the lamp to warm before resuming operations 7 Record the new lamp s serial...

Page 155: ...isplay keypad 3 Press 1 Change lamp Tip Be sure to enter the lamp s serial number and not its part number when performing this procedure 4 Specify the serial number of the new lamp in the active field...

Page 156: ...fective when the detector fails to power on the display is blank the fans do not operate To replace the fuses Replace both fuses even when only one is open or otherwise defective 1 Power off the detec...

Page 157: ...he new fuses suit your requirements 5 Insert the new fuses into the fuse holder 6 Insert the fuse holder into the receptacle and gently push until it locks into position on the rear panel 7 Start the...

Page 158: ...4 16 Maintenance Procedures...

Page 159: ...el can access the service diagnostics Error messages Startup calibration and other error messages and recommended actions for correcting the errors Diagnostic tests User diagnostic tests for troublesh...

Page 160: ...rifying the detector on page 3 34 and Manual wavelength calibration on page 3 35 have verification and calibration information and procedures Startup error messages Error message Description Correctiv...

Page 161: ...n the home screen If a catastrophic error appears Lamp external input conflict A timed event or front panel action attempts to change the lamp state in conflict with enabled lamp input contact closure...

Page 162: ...ce A D A D communication test failed 1 Power off and power on again 2 If the problem persists call Waters Technical Service Communication failure Sample A D A D communication test failed 1 Power off a...

Page 163: ...persists power off and power on again 3 If the problem persists call Waters Technical Service Filter initialization failure Order filter position Unit sensors cannot find the order filter position 1 E...

Page 164: ...on again 3 If the problem persists call Waters Technical Service Filter initialization failure Shutter position Unit sensors cannot find the shutter position 1 Ensure solvent is flowing 2 If the prob...

Page 165: ...tch is open 1 Close the lamp door 2 Remove any cooling vent obstructions 3 If the problem persists call Waters Technical Service Method not found Stored method data is not valid Power off and power on...

Page 166: ...form the tests and change settings 1 Press DIAG on the keypad The Diagnostics list appears System cannot respond Error occurs while unit is positioning next wavelength or changing modes during initial...

Page 167: ...sticky diagnostic test is active the home screen displays a wrench icon If no tests are active the wrench icon does not appear on the home screen Home screen showing active sticky diagnostic settings...

Page 168: ...100 EU using a standard clean water reference 2 Raman S N Test Runs the 15 minute signal to noise test for water 3 Auto Optimize Gain Displays a table of recommended gain settings for a method based...

Page 169: ...urrent sample and reference energy readings appear in energy units from 1 to 10 000 Sample and reference energy tests To perform sample and reference energy tests 1 Press DIAG then press 5 Sample ref...

Page 170: ...you start the signal to noise test the detector sets the excitation wavelength to 350 nm it sets the emission wavelength and gain to the values stored during the normalize units test Clean degassed wa...

Page 171: ...nput output list press 1 Auto zero offsets Auto zero offsets screen 2 Select Cancel Shift 0 if you want to clear the offset on both channels to zero Setting the fixed EU value This function sets the v...

Page 172: ...Input output list press 3 Fix voltage to select a voltage for the analog output You can select a voltage for both output channels range 0 10 to 1 10 V Fix voltage screen Monitoring contact closures a...

Page 173: ...to OFF or vice versa c Press Enter to select the second switch Change lamp function Whenever you change the lamp use this function to enter a new serial number and installation date See page 4 12 and...

Page 174: ...and then press Enter Testing the keypad To test the keypad 1 Press DIAG and then press 7 Lamp display keypad 2 Press 2 Test keypad Keypad test 3 Press any key to begin the test and then press each ke...

Page 175: ...ttings screen provides three additional diagnostic functions Generate test peaks Generates test peaks to calibrate a chart recorder or data system Manually override the optical filter Selects a filter...

Page 176: ...The gain is automatically set to 1000 Generate test peaks message Tip You must manually disable the Generate test peaks function to stop it 3 Press 1 Disable test peaks to stop generating test peaks O...

Page 177: ...ng the available linear gain range of 1 to 1000 This feature is intended for use when samples or mobile phase solvents exhibit high fluorescence emissions that saturate the PMT signal but reducing con...

Page 178: ...uments as well as with the detector Most detector problems are relatively easy to correct If you are unable to correct a problem or failed condition after running the user diagnostics applicable to th...

Page 179: ...led General hardware troubleshooting on page 5 21 describe error messages that can appear onscreen as you start up or operate the detector and it suggests corrective actions Power surges Power surges...

Page 180: ...nd if necessary replace fuse s Bad LCD or control board Call Waters Technical Service Front panel displays odd characters Faulty EPROMs or bad LCD control board Call Waters Technical Service Keypad no...

Page 181: ...nd if necessary replace the RS 232 cable Xenon lamp does not light Faulty lamp Replace the lamp Lamp not plugged in Plug in the lamp connector Bad lamp power supply Contact Waters Technical Service La...

Page 182: ...5 24 Error Messages Diagnostic Tests and Troubleshooting...

Page 183: ...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 184: ...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 ho...

Page 185: ...ing Risk of explosion Warning Risk of eye injury Warning Pressurized nonmetallic or polymer tubing can burst Observe these precautions when working around such tubing Wear eye protection Extinguish al...

Page 186: ...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 syst...

Page 187: ...nisms 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 s...

Page 188: ...ler le droit de l utilisateur exploiter l quipement Achtung Jedwede nderungen oder Modifikationen an dem Ger t ohne die ausdr ckliche Genehmigung der f r die ordnungsgem e Funktionst chtigkeit verantw...

Page 189: ...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 190: ...iamenti 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 usuari...

Page 191: ...Warnings that apply to all Waters instruments A 9 Nonmetallic Tetrahydrofuran THF Methylene chloride Dimethyl sulfoxide THF...

Page 192: ...tueuses Vorsicht Der Benutzer wird darauf aufmerksam gemacht dass bei unsachgem er Verwenddung des Ger tes die eingebauten Sicherheitseinrichtungen unter Umst nden nicht ordnungsgem funktionieren Atte...

Page 193: ...l instrument Vorsicht Zum Schutz gegen Feuer die Sicherungen nur mit Sicherungen ersetzen deren Typ und Nennwert auf den Tafeln neben den Sicherungsabdeckungen des Ger ts gedruckt sind Attenzione per...

Page 194: ...e 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 terminal F...

Page 195: ...ng symbols A 13 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...

Page 196: ...A 14 Safety Advisories...

Page 197: ...ecification Operating temperature 4 to 40 C 39 2 to 104 F Operating humidity 20 to 80 noncondensing Shipping and storage temperature 40 to 70 C 40 to 158 F Shipping and storage humidity 20 to 80 nonco...

Page 198: ...this grounding system is connected to the third pin ground pin on the electrical power cord plug b Overvoltage Category II Pertains to instruments that receive their electrical power from a local lev...

Page 199: ...to 5 0 seconds Hamming default 0 1 to 99 seconds RC Multichannel 1 to 50 seconds Hamming default 1 to 99 seconds RC Optical Component Specifications Lamp source Xenon arc lamp 150 W Flow cell Axial Il...

Page 200: ...B 4 Specifications...

Page 201: ...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 Wavelength selection...

Page 202: ...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 F...

Page 203: ...re 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 temperature applicati...

Page 204: ...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...

Page 205: ...or their critical solution temperature is above 75 C Some solvents prove immiscible with solvents at either end of the lipophilicity scale These solvents receive a dual M number The first number alwa...

Page 206: ...duled to be more than one day Use a minimum of 15 mL for sparge equipped units and a minimum of 45 mL for inline vacuum degasser equipped units Some modern systems such as Waters Alliance may require...

Page 207: ...lubility 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 appl...

Page 208: ...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 equil...

Page 209: ...w 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 dega...

Page 210: ...tropropane 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 Methano...

Page 211: ...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 BRIJ 35 0 1 190 Sodium formate 10 mM 200 CHA...

Page 212: ...en sample scanning over a range of wavelengths can be necessary to determine the best wavelength for a particular analysis 1 Willard H H and others Instrumental Methods of Analysis 6th ed Litton Educa...

Page 213: ...lder 12 Allene 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 Eth...

Page 214: ...C 14 Solvent Considerations...

Page 215: ...al 3 22 other parameters 3 20 signal adjusting 3 20 single wavelength signal 3 22 single channel 3 20 analog signals 2 6 anthracene 3 53 3 64 audience and purpose iv Auto Zero key 3 12 automatic secon...

Page 216: ...r connecting 2 28 2 29 charting difference plot 3 20 MaxPlot function 3 20 chemical hazard warning A 5 chemiluminescence 1 2 chromatography fluorescence 1 2 cleaning the flow cell 4 5 cleaning detecto...

Page 217: ...ns C 9 deleting a timed event 3 47 design electronic 1 9 optical 1 9 detector exterior cleaning 4 15 overview 1 7 setup 2 2 DIAG key 3 13 diagnostic settings auto zero offset 5 10 5 13 contact closure...

Page 218: ...excitation 1 3 energy units 1 6 3 19 Enter key 3 15 entering negative numbers 3 15 entrance slits 1 9 environmental specifications 2 2 B 1 equipment guidelines iv A 6 erbium filter 1 8 scan 3 53 erro...

Page 219: ...hold timed event parameter 3 46 trace 3 13 Fluorescence screen display 3 11 icons 3 5 flushing flow cell 4 5 front left hand panel cover 4 4 functions analog outputs single channel 3 20 auto zero on w...

Page 220: ...ests 5 10 5 12 inputs disabling 3 16 enabling 3 16 signals 2 11 inspecting flow cells 4 5 installation columns 2 3 network guidelines 2 8 2 21 installing a new lamp 4 12 intended use v inverting the c...

Page 221: ...stics 3 14 warranty 4 13 Lamp key 3 14 lamp off icon 3 7 lamp on icon 3 6 lamp on off connections to 600 Series Pump 2 30 light filters 1 3 light sources excitation 1 3 line spikes 5 21 local remote c...

Page 222: ...xt arrow 3 13 Next icon 3 8 Next key 3 13 noise adjusting filter 3 20 filters 3 19 specifications B 3 noise filtering 1 12 normalized units of fluorescence 1 5 numerical keys 3 14 O obtaining MaxPlot...

Page 223: ...s B 2 performing verification procedures 3 34 personality board 1 13 photomultiplier tube 1 4 1 9 calibrating 1 12 gain setting 1 17 sensitivity 1 12 physical specifications B 1 plus minus key 3 15 PM...

Page 224: ...maintenance 4 2 sample and reference energy diagnostic tests 5 10 5 11 sample energy 3 56 sample scan procedure 3 55 3 56 screens 3 55 when to run 3 56 sample exciting 1 4 Scale key 3 13 3 23 scale z...

Page 225: ...entrance 1 9 slits exit 1 9 solvent buffered solvents C 6 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 overview C 8 specif...

Page 226: ...operation 1 1 threshold events clearing 3 51 programming 3 47 tick marks generating 3 53 time constant changing 3 16 function 3 20 parameter 3 22 timed event parameter 3 45 timed events and methods 3...

Page 227: ...ions 2 31 chart mark connections 2 31 configuring the detector lamp signal 2 30 connecting 2 29 2 33 inject start connections 2 32 lamp on off connections 2 30 Waters 717plus Autosampler auto zero con...

Page 228: ...Index 14...

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