Waters 2996 Operator'S Manual Download Page 9

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Caution, risk of electric shock (high voltage)

Attention, risque de commotion électrique (haute tension)

Vorsicht, Elektroschockgefahr (Hochspannung)

Precauzione, rischio di scossa elettrica (alta tensione)

Precaución, peligro de descarga eléctrica (alta tensión)

Caution, risk of needle-stick puncture

Attention, risques de perforation de la taille d’une aiguille

Vorsicht, Gefahr einer Spritzenpunktierung

Precauzione, rischio di puntura con ago

Precaución, riesgo de punción con aguja

Caution, ultraviolet light

Attention, rayonnement ultrviolet

Vorsicht, Ultraviolettes Licht

Precauzione, luce ultravioletta

Precaución, emisiones de luz ultravioleta

Commonly Used Symbols

(Continued)

Summary of Contents for 2996

Page 1: ...Waters 2996 PDA Detector Operator s Guide 34 Maple Street Milford MA 01757 71500023202 Revision C...

Page 2: ...or consequential damages in connection with or arising from the use of this document 2001 WATERS CORPORATION PRINTED IN THE UNITED STATES OF AMERICA ALL RIGHTS RESERVED THIS DOCUMENT OR PARTS THEREOF...

Page 3: ...oid the user s authority to operate the equipment Important Toute modification sur cette unit n ayant pas t express ment approuv e par l autorit responsable de la conformit la r glementation peut annu...

Page 4: ...trouvant proximit N utilisez pas de tuyau de Tefzel fortement ab m ou d form N utilisez pas de tuyau de Tefzel avec de l acide sulfurique ou nitrique ou du t trahydrofurane THT Sachez que le chlorure...

Page 5: ...dimetilsolfossido provocano rigonfiamento nei tubi Tefzel che riducono notevolmente il limite di pressione di rottura dei tubi stessi Advertencia manipular con precauci n los tubos de polimero bajo pr...

Page 6: ...la protezione fornita dall apparecchiatura potr essere invalidata Advertencia El usuario deber saber que si el equipo se utiliza de forma distinta a la especificada por el fabricante las medidas de p...

Page 7: ...d effectuer la maintenance de l instrument Vorsicht Zur Vermeidung von Stromschl gen sollte das Ger t vor der Wartung abgeschaltet und vom Netz getrennt werden Precauzione per evitare il rischio di sc...

Page 8: ...terminal Borne du cadre ou du ch ssis Rahmen oder Chassisanschluss Terminale di struttura o telaio Borne de la estructura o del chasis Caution or refer to manual Attention ou reportez vous au guide V...

Page 9: ...ica alta tensi n Caution risk of needle stick puncture Attention risques de perforation de la taille d une aiguille Vorsicht Gefahr einer Spritzenpunktierung Precauzione rischio di puntura con ago Pre...

Page 10: ...ling CDC NIH Manual 1984 Calibration Follow acceptable methods of calibration with pure standards to calibrate methods Use a minimum of five standards to generate a standard curve The concentration ra...

Page 11: ...Connecting Analog Output Cables 6 1 4 2 Connecting Event Cables 7 1 5 Fluidic Connections 9 1 6 Starting Up and Shutting Down the Detector 11 Chapter 2 Diagnostics and Calibration 15 2 1 Diagnostics 1...

Page 12: ...ta Points 35 4 4 1 Calculating Absorbance 35 4 4 2 Resolution 38 4 4 3 Filtering Data 38 Chapter 5 Spectral Contrast Theory 41 5 1 Comparing Absorbance Spectra 41 5 2 Representing Spectra as Vectors 4...

Page 13: ...Table of Contents xiii Appendix A Detector Specifications 51 Appendix B Spare Parts 53 Appendix C Mobile Phase Absorbance 55 Index 59...

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

Page 15: ...ling the Flow Cell 23 3 4 Lamp Power Connector and Mounting Screws 26 3 5 Fuse Block 27 4 1 Optics Assembly Light Path 30 4 2 Benzene Spectrum at 1 2 nm Resolution 32 4 3 Photodiodes Discharged by Lig...

Page 16: ...xvi List of Figures...

Page 17: ...1 3 Event Out Terminal Specifications on Contact Closure 9 1 4 LED Indicator Sequences During Startup 12 2 1 2996 Detector Troubleshooting 15 4 1 Optics Assembly Components 30 A 1 2996 Detector Speci...

Page 18: ...xviii List of Tables...

Page 19: ...l and set up the 2996 Detector Chapter 2 describes how to troubleshoot the 2996 Detector Chapter 3 describes how to replace the flow cell the lamp and the fuse Chapter 4 explains the principles involv...

Page 20: ...nts as part of the Millennium 32 system Also covers the installation configuration and use of acquisition servers such as the LAC E32 module the busLAC E card and interface cards used to communicate w...

Page 21: ...at more of the same type of item can optionally follow For example You can store filename1 filename2 in each folder A right arrow between menu options indicates you should choose each option in sequen...

Page 22: ...xxii...

Page 23: ...t are not compatible with Millennium32 or MassLynx software control 1 1 Installation Site Requirements Install the 2996 PDA Detector at a site that meets the specifications in Table 1 1 and Figure 1 1...

Page 24: ...requires no voltage adjustment The electrical power requirements for the Waters 2996 PDA Detector are Voltage range 100 to 240 Vac Total power 100 VA Frequency range 50 to 60 Hz Fuses The 2996 PDA De...

Page 25: ...er end to a power outlet Figure 1 2 Detector Rear Panel 1 3 Millennium32 Workstation Connections The 2996 Detector requires signal connections to the Millennium 32 Workstation over the IEEE 488 bus Al...

Page 26: ...rd on the workstation is not important For example you can connect the 2690 Separations Module before or after the 2996 Detector 2 Use another IEEE 488 cable to connect to the stackable connector on t...

Page 27: ...g the IEEE 488 Address To set the IEEE 488 address for the 2996 Detector Use a small screwdriver or similar device to set the IEEE 488 switches on the detector rear panel Figure 1 4 to the IEEE 488 ad...

Page 28: ...the Millennium 32 Help Required Materials One small flat blade screwdriver One electrical insulation stripping tool Analog signal cables included with the Waters 2996 Detector Startup Kit Procedure To...

Page 29: ...enerally used for inject start Two output programmable event table signal terminals If an inject start signal is not available over the IEEE 488 bus you must provide a signal at Event In 1 terminal on...

Page 30: ...eps 1 Insert the bare wires at one end of the event signal cable into the positive and negative slots of the appropriate event input or output terminal Figure 1 6 Tighten the two screws to secure the...

Page 31: ...Low trigger 1 8 V High trigger 3 0 V Protected to 30 V Minimum pulse width 30 msec this may limit compatibility with valves that require a quick pulse Maximum current 5 mA Table 1 3 Event Out Terminal...

Page 32: ...east 1 to 2 feet 30 to 60 cm to prevent air bubbles from forming in the flow cell 2 Cut the two lengths of tubing as follows a Use a Waters 1 16 inch stainless steel tubing cutter or a file with a cut...

Page 33: ...in the waste container 1 6 Starting Up and Shutting Down the Detector The entire startup procedure takes less than 1 minute Once completed you should allow the 2996 Detector to warm up for at least 1...

Page 34: ...anel of the detector Figure 1 8 If both lights remain illuminated the detector passed the internal diagnostics If either indicator light blinks or is off refer to the troubleshooting tables in Chapter...

Page 35: ...of the detector to the 0 Off position OFF FLASHING The 2996 is running power on self tests FLASHING FLASHING 2996 has failed one of the self tests Contact Waters Technical Service OFF FLASHING The 299...

Page 36: ...14 Installation 1...

Page 37: ...w accessed from Run Samples in the Millennium 32 software If you encounter a problem that you cannot troubleshoot Section 2 1 Diagnostics contact Waters Technical Service at 800 252 4752 U S and Canad...

Page 38: ...ctor waste outlet Weak lamp Replace lamp Section 3 1 2 Removing the Flow Cell Shutter failure message Shutter failed Run the Shutter diagnostic For details see Millennium 32 Help Detector not respondi...

Page 39: ...cs from Run Samples in the Millennium 32 software For more information on Run Samples and PDA diagnostics refer to the Millennium 32 Help Change in reference spectrum Mobile phase contains gas or is c...

Page 40: ...rate to set the 486 nm peak at the proper wavelength Ensure precise data for library matching Note The system administrator can restrict access to the PDA Calibration window by disabling access to Run...

Page 41: ...ow fails and the lamp status light is on Table 2 1 The 2996 Detector causes high backpressure Note Conditions other than a dirty flow cell may cause decreased lamp intensity For more information refer...

Page 42: ...n then run the pump for 10 minutes 3 Test the lamp energy by performing the Lamp diagnostic test For details refer to the Millennium 32 Help If the lamp diagnostic fails and the lamp has not been used...

Page 43: ...he Flow Cell Assembly 6 Use the Phillips screwdriver to loosen the three thumbscrews that hold the flow cell assembly to the optics bench and the thumbscrew that secures the bracket holding the fluidi...

Page 44: ...surface finish and the alignment of the lenses are critical to the performance of the 2996 Detector Be careful not to touch or damage the lenses and the lens holders STOP Attention To prevent lens co...

Page 45: ...he Flow Cell 2 Use the small flat blade screwdriver to gently pry the lens assembly from the flow cell body at the slots 3 Use a lens tissue or a nonparticulating swab to wipe the lens with methanol S...

Page 46: ...Tighten until the screws meet the flow cell disk then tighten each screw 1 4 turn If a torque screwdriver is available tighten the screws to 16 in oz 0 113 N m 6 Check for leaks If you find any leaks...

Page 47: ...mobile phase also affects the apparent lamp intensity For example acetonitrile is more transparent than methanol at wavelengths less than 220 nm Caution To avoid electrical hazards and exposure to UV...

Page 48: ...ay from the chassis 3 Open lamp secure panel Figure 3 4 Lamp Power Connector and Mounting Screws Caution To avoid electrical hazards when you perform the following procedure power off the 2996 Detecto...

Page 49: ...Reconnect the lamp power connector Figure 3 4 10 Secure the lamp access panel 11 Install the front panel cover 12 Reconnect the power cord and power on the 2996 Detector 3 3 Replacing the Fuses Replac...

Page 50: ...ock while you pull out the block 4 Remove the fuses from the block then install the new fuses 5 Position the fuse block with the small tab pointing down then push in the block until the side clips eng...

Page 51: ...of operation of the optics and electronics of the Waters 2996 PDA Detector 4 1 2996 Detector Optics The 2996 Detector is an ultraviolet visible light UV Vis spectrophotometer with 512 photodiodes Opt...

Page 52: ...back to a reference diode which measures the intensity of the light emitted by the lamp The detector uses this measurement to keep the lamp output constant Flow cell assembly Houses the segment of the...

Page 53: ...mirror and mask The mirror focuses light transmitted through the flow cell onto the aperture at the entrance to the spectrographic portion of the optics The mirror mask defines the beam of light focus...

Page 54: ...t of light striking the photodiode array to determine the absorbance of the sample in the flow cell The array consists of 512 photodiodes arranged in a row Each photodiode acts as a capacitor by holdi...

Page 55: ...s of an individual diode is the exposure time The 2996 Detector requires less than 10 msec to sequentially read all of the diodes in the array and process the data The minimum exposure time is 10 msec...

Page 56: ...terium light source from 190 to 800 nm To minimize detector noise Auto Exposure adjusts the exposure time from 50 to 90 of full scale The Auto Exposure time setting ensures that the photodiodes are No...

Page 57: ...hs of interest and over which the mobile phase absorbs minimally Appendix C Mobile Phase Absorbance Setting the bandwidth to higher values can improve signal to noise ratios 4 4 Computing Absorbance D...

Page 58: ...eer s Law applies only to well equilibrated dilute solutions It assumes that the refractive index of the sample remains constant that the light is monochromatic and that no stray light reaches the det...

Page 59: ...ely after the dark current measurement and before any components are eluted the 2996 Detector records a reference spectrum The reference spectrum is a measure of lamp intensity and mobile phase absorb...

Page 60: ...ple Rate Sample rate is the number of data points per second reported to the Millennium 32 database The number of times the photodiodes are read during the sample rate interval is dependent on the exp...

Page 61: ...a good signal to noise ratio for most chromatographic separations Note the following with regard to filtering data The noise filter is a digital low pass filter The filter calculates a data point tha...

Page 62: ...40 Principles of the 2996 PDA Detector Optics 4...

Page 63: ...f multiple compounds in the same peaks coelution or due to nonideal conditions such as noise photometric error or solvent effects 5 1 Comparing Absorbance Spectra The shape of an absorbance spectrum i...

Page 64: ...centration for peaks that are less than 1 0 AU across the collected wavelength range Vector direction contributes to the identification of a compound since the direction is a function of the absorbanc...

Page 65: ...the spectrum of Compound B The vector for Compound B points in a direction different from that of the vector for Compound A The difference in direction which reflects the difference in the absorbance...

Page 66: ...at have different shapes have vectors that point in different directions The angle between the two vectors of any two spectra the Spectral Contrast angle quantifies the magnitude of the shape differen...

Page 67: ...ctral Contrast Angle Spectra with Similar Shapes In Figure 5 4 the absorbance spectra of two compounds A and B are similar and therefore have a small Spectral Contrast angle 3 0 Normalized Absorbance...

Page 68: ...may exhibit slight differences because of detector noise photometric error high sample concentration or variations in solvent conditions The spectra in Figure 5 5 for example show how instrument nois...

Page 69: ...ogeneity You can assess the significance of spectral inhomogeneity by comparing a Spectral Contrast angle to a Threshold angle Section 5 4 4 5 4 1 Detector Noise Statistical and thermal variations add...

Page 70: ...d composition do not change isocratic operation the background absorbance if any by the solvent remains constant A change however in solvent pH or composition such as occurs in gradient operation can...

Page 71: ...Effects of pH and Solvent Concentration on the Absorbance Spectrum of p Aminobenzoic Acid Wavelength nm Absorbance Absorbance Wavelength nm pH 6 9 pH 5 1 pH 3 1 Effect of Concentration Effect of pH No...

Page 72: ...50 Spectral Contrast Theory 5...

Page 73: ...to 800 nm Wavelength accuracy 1 nm Linearity range Per ASTM 685 79 5 at 2 0 AU propylparaben at 256 nm Spectral resolution 1 2 nm Baseline noise 1 5 10 5 AU peak to peak dry at 254 nm Drift 1 10 3 AU...

Page 74: ...52 Detector Specifications A...

Page 75: ...7919 Flow cell semi preparative WAT057463 Flow cell microbore WAT057462 Flow cell inert WAT057461 Flow cell Autopurification 289000612 Variable pathlength flow cell WAT057664 Gasket flow cell 2 WAT057...

Page 76: ...54 Spare Parts B...

Page 77: ...that is autozeroed out Wavelength pH and concentration of the mobile phase will affect its absorbance Examples of several mobile phases are provided in Table C 1 Table C 1 Mobile Phase Absorbance Meas...

Page 78: ...2 42 2 50 2 45 2 37 1 96 0 50 0 12 0 04 0 01 Buffers and Salts Ammonium acetate 10 mM 1 88 0 94 0 53 0 29 0 15 0 02 0 01 Ammonium bicarbonate 10 mM 0 41 0 10 0 01 0 01 EDTA disodium 1 mM 0 11 0 07 0 0...

Page 79: ...citrate 10 mM 2 48 2 84 2 31 2 02 1 49 0 54 0 12 0 03 0 02 0 01 Sodium formate 10 mM 1 00 0 73 0 53 0 33 0 20 0 03 0 01 Sodium phosphate 100 mM pH 6 8 1 99 0 75 0 19 0 06 0 02 0 01 0 01 0 01 0 01 0 01...

Page 80: ...02 0 02 0 02 0 02 0 01 Detergents BRIJ 35 1 0 06 0 03 0 02 0 02 0 02 0 01 0 01 CHAPS 0 1 2 40 2 32 1 48 0 80 0 40 0 08 0 04 0 02 0 02 0 01 SDS 0 1 0 02 0 01 0 01 Triton X 100 0 1 2 48 2 50 2 43 2 42...

Page 81: ...n IEEE 488 6 rear panel 3 terminal strip 8 Contact closures 6 Contacting Waters Technical Service 15 17 Conventions documentation xx D Dark current 37 Data acquisition Auto Exposure parameter 34 Expos...

Page 82: ...lengths 55 N Noise effects 47 Nonidealities 47 Non IEEE 488 connections 6 O Outputs 6 8 9 P Parts spare 53 Photodiode array 32 Photometric error 48 Power connections 2 Purity Angle photometric error e...

Page 83: ...multiple wavelengths 44 derived from two wavelengths 43 spectra representing 42 spectral contrast 42 W Waters 2996 absorbance calculations 35 37 aperture width 31 dark current 37 detector optics over...

Page 84: ...62 Index I N D E X...

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