Waters 2998 Operator'S Manual Download Page 11

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Table of Contents

Wavelength selection for chromophore detection......................................... C-13
Mobile phase absorbance............................................................................... C-14

Index ..................................................................................................... Index-1

Summary of Contents for 2998

Page 2: ...bility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Waters Corporation be liable for incidental or...

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: ...in this section Applicable symbols Audience and purpose This guide is intended for personnel who install operate and maintain the Waters 2998 Photodiode Array PDA detector Symbol Definition Manufactur...

Page 5: ...m for calibration instructions Quality control Routinely run three QC samples that represent subnormal normal and above normal levels of a compound Ensure that QC sample results fall within an accepta...

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: ...ity control v ISM classification v ISM Classification ISM Group 1 Class B v EC authorized representative vi 1 2998 PDA Detector Optics Principles 1 1 Detector optics 1 2 Calculating absorbance 1 3 Flo...

Page 8: ...e Ethernet cable 2 6 Network installation guidelines 2 7 Connecting to other instruments 2 8 Plumbing the detector 2 16 Making the gas supply connection 2 18 Starting up and shutting down the detector...

Page 9: ...ear panel interface connections 4 5 General troubleshooting 4 6 Power surges 4 6 Clearing bubbles from the flow cell 4 7 Detector troubleshooting 4 7 5 Spectral Contrast Theory 5 1 Comparing absorbanc...

Page 10: ...eparation checklist C 2 Water C 2 Using buffers C 2 Tetrahydrofuran C 3 Solvent miscibility C 3 How to use miscibility numbers C 5 Buffered solvents C 6 Head height C 6 Minimum tubing bend radius reco...

Page 11: ...Table of Contents xi Wavelength selection for chromophore detection C 13 Mobile phase absorbance C 14 Index Index 1...

Page 12: ...xii Table of Contents...

Page 13: ...ou should understand the principles that underlie operation of the detector s optics and electronics Contents Topic Page Detector optics 1 2 Flow cell operating principles 1 4 Resolving spectral data...

Page 14: ...0 to 800 nm The figure below illustrates the light path through the optics assembly of the detector Optics assembly light path The following table describes the optics assembly components Optics assem...

Page 15: ...g eluent and sample through which the polychromatic light beam passes Spectrograph mirror and mask The mirror focuses light transmitted through the flow cell onto the slit at the entrance to the spect...

Page 16: ...nt As concentration increases the chemical and instrumental requirements of Beer s law are sometimes violated resulting in a deviation from absorbance versus concentration linearity The absorbance of...

Page 17: ...hotodiode spacing the detector s 50 m slit determines the intensity and bandwidth of the light that strikes the photodiode array Reducing the bandwidth increases the resolving power of the detector As...

Page 18: ...odiode array to determine the absorbance of the sample in the flow cell The array consists of a row of 512 photodiodes Each photodiode acts as a capacitor initially holding a fixed amount of charge Li...

Page 19: ...only minimally see Appendix C You can further improve the signal to noise ratio by increasing the bandwidth parameter For example you can choose to operate at 3 6 nm instead of at 1 2 nm resolution S...

Page 20: ...lick anywhere in the table and then click Table Properties Click the Columns tab and then scroll down to find the Points Across Peak field Clear the check box and then click OK If the Points Across Pe...

Page 21: ...take a dark current reading for each diode After updating the detector opens the shutter and then subtracts the dark current values as shown in the above equation Reference spectrum The reference spe...

Page 22: ...eak purity analysis set the 3D resolution to 1 2 nm For chromatograms 2D data select a resolution to optimize signal amplitude baseline noise and linear dynamic range When the monitoring wavelength fo...

Page 23: ...te interval depends on its exposure time For example if exposure time is 25 msec and sample rate is 20 Hz then exposures per sample is 3 20 3 30 3 40 3 50 3 60 3 70 3 80 3 90 4 00 4 10 4 20 0 00 0 05...

Page 24: ...online Help you can apply an optional noise filter via the Digital Filtering parameter to the data acquired The following table lists the digital filter settings for the allowable data rates Tip For...

Page 25: ...at you select It then subtracts that value from the absorbance value Abs Comp t Abs t CRef t where Abs Comp absorbance compensated Abs absorbance CRef compensation reference t elapsed time after start...

Page 26: ...1 14 2998 PDA Detector Optics Principles...

Page 27: ...up the detector Contents Topic Page Before you begin 2 2 Unpacking and inspecting 2 2 Laboratory site selection 2 3 Connecting to the electricity source 2 4 Making signal connections 2 5 Plumbing the...

Page 28: ...s against the packing lists to ensure that you received all items 2 Save the shipping cartons for future transport or shipment If you discover any damage or discrepancy when you inspect the contents o...

Page 29: ...on page 2 4 indicate that the detector is a stackable unit and does not require extra bench space Place the detector close to the outlet of the column to minimize band broadening which will reduce the...

Page 30: ...nd connection in the electrical outlet must be common and connected near the system Warning Avoid electrical shock Use power cord SVT type in the United States and HAR type or better in Europe For oth...

Page 31: ...e detector on at this time Making signal connections See also Waters Ethernet Instrument Getting Started Guide The following figure shows the rear panel location of the connectors used to operate the...

Page 32: ...on In a single Waters instrument system configuration the connection hardware requires only one standard Ethernet cable provided in the startup kit Single Waters instrument connection Multiple Waters...

Page 33: ...hubs are not supported Waters Ethernet instrument connections Making inject start signal connections When you are using an Ethernet data system with the detector the data system or controller must re...

Page 34: ...ector as shown in the following table For information on injection trigger signals from a manual injector see page 2 15 Connecting to other instruments This section describes signal connections betwee...

Page 35: ...you connect the shield of the cable to ground at one instrument only To connect the detector to other instruments use the two analog out event in I O connectors and their mating connectors on the rear...

Page 36: ...thod to the detector using the lamp button or rebooting the detector Chart mark Adds a chart mark at 10 of full scale to either or both analog output channels Signal Out 1 and Signal Out 2 and is conf...

Page 37: ...PDA detector connector A Pin 1 inject start red Pin 1 inject start red Pin 2 inject start black Pin 2 inject start black Warning Fire and explosion hazard Failure to connect the stop flow output could...

Page 38: ...ule and the detector Detector connections to an Alliance separations module Alliance separations module connector B 2998 PDA detector connector B Pin 4 stop flow red Pin 6 switch 1 red Pin 5 stop flow...

Page 39: ...Analog output connections to a chart recorder Analog output connections to a chart recorder Chart recorder connectors 2998 PDA detector connector B Pen 1 Pin 1 signal out red Pen 1 Pin 2 signal out bl...

Page 40: ...le make the connections shown in the following table and figure Analog output connections to the eSAT IN module Detector connections to the eSAT IN module SAT IN module connector 2998 PDA detector con...

Page 41: ...inject start function To send an auto zero or chart mark signal from an injector to the detector make the connections shown in the following tables and figures Tip The firmware defaults to auto zero...

Page 42: ...adening 2 Measure the length of tubing needed to connect the detector outlet to a waste collection bottle Tip Ensure the length of this tubing is at least 30 to 60 cm 1 to 2 feet to prevent air bubble...

Page 43: ...of the column outlet tubing in the fitting of the column outlet finger tighten the reusable finger tight fitting until it is snug and then add a quarter turn 6 Seat the other end of the tubing in the...

Page 44: ...f the nitrogen fitting on the rear panel of the detector Starting up and shutting down the detector The detector startup procedure takes less than one minute Once completed you should allow the detect...

Page 45: ...gy diagnostic test Ensure that the priming solvent is correctly composed and that it is of high quality and miscible with any other solvents used in your system Use filters in all solvent reservoirs a...

Page 46: ...Power LED The power LED on the detector s front panel indicates when the instrument is powered on or powered off It is steady green when the instrument is working properly Lamp LED The lamp LED to the...

Page 47: ...ification The detector uses a standard 10 mm path length spectrophotometric cell quartz cuvette You insert the cuvette with a frosted side facing up in the cuvette holder which you then place in the d...

Page 48: ...under identical flow cell conditions Before you begin Recommendation To ensure accurate results use a 10 mm path length quartz cuvette and matched pairs from the same manufacturing lot of quartz cuve...

Page 49: ...figure on page 2 22 Recommendations Ensure that you have enough liquid 3 mL in the cuvette so that when it is inserted into the holder you can see liquid through the cuvette holder aperture that is t...

Page 50: ...aintain optimum system performance replace the front left panel cover before resuming normal operation of the detector 6 Insert a reference cuvette containing the mobile phase standard and run a zero...

Page 51: ...routine maintenance procedures you should perform on the detector Contents Topic Page Contacting Waters technical service 3 2 Maintenance considerations 3 2 Routine Maintenance 3 3 Maintaining the flo...

Page 52: ...re of the symptom Instrument serial number Solvent s Method parameters sensitivity and wavelength Type and serial number of column s Sample type Empower or MassLynx software version and serial number...

Page 53: ...ease baseline noise 3 Flush buffered mobile phases out of the detector with HPLC grade water followed by a 5 to 10 methanol solution each time the detector is shut down This process prevents Plugging...

Page 54: ...bly Flushing the flow cell Required Materials HPLC grade water HPLC grade methanol If the flow cell requires cleaning first try flushing it with solvent To flush the flow cell 1 Select a solvent compa...

Page 55: ...flow 3 Lift up the detector front cover and pull the front cover from the detector chassis 4 Disconnect the detector s inlet tubing from the column outlet connection 2998 PDA detector analytical flow...

Page 56: ...h Note that the flow cell uses the alignment pins on the optics bench 8 Gently push the front of the assembly until it seats on the front alignment pins Installing the analytical flow cell assembly 9...

Page 57: ...from the date of purchase whichever comes first To remove the lamp 1 Power off the lamp 2 Power off the detector and disconnect the power cable from the rear panel 3 Allow the lamp to cool for 30 minu...

Page 58: ...pressure To prevent shattering the glass use care when disposing of the lamp Waters suggests that you adequately cushion an old lamp by containing it in the packaging of its replacement before you di...

Page 59: ...then reconnect the lamp power connector 5 Replace the front panel cover 6 Power on the detector and then wait 1 hour for the lamp to warm before resuming operations Tip Cycling power to the detector...

Page 60: ...operate To replace the fuses Requirement Replace both fuses even when only one is open or otherwise defective 1 Power off the detector and disconnect the power cord from the power entry module 2 Pinch...

Page 61: ...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 until the assembly locks into position 6 Reco...

Page 62: ...3 12 Maintaining the Detector...

Page 63: ...e 2998 PDA detector However bear in mind that the detector measures only the bulk properties of a system Therefore the cause of an apparent detector problem may actually originate with the chromatogra...

Page 64: ...e flow cell with solvent and remove any air Recommendation Impurities in the flow cell may affect wavelength verification results Ensure that the flow cell is clean before you calibrate To verify dete...

Page 65: ...mmendation Impurities in the flow cell may affect wavelength calibration Ensure that the flow cell is clean before you perform calibration Before you begin this procedure your detector must be set up...

Page 66: ...ections or contact closures on the rear panel of the 2998 PDA detector This display gives you a real time status of the instrument s signal connections A green LED symbol indicates a signal cable is c...

Page 67: ...output signal for the selected signal connection indicates whether it is opened or closed PDA detector analog out event in connections Signal connections Description Analog 1 Out Method programmable...

Page 68: ...ne spikes and transient energy sources can adversely affect detector operations Be sure that the electrical supply used for the detector is properly grounded and free from any of these conditions Powe...

Page 69: ...he detector Information regarding the error that caused the failure can be found in the console log See also 2998 PDA Detector online Help Constant red Indicates a detector failure that prevents furth...

Page 70: ...of 0 009 inch 0 23 mm ID tubing connected to the detector waste outlet Continuous audible tone Detector failure Power the PDA detector off and on again Detector not responding to console Bad or disco...

Page 71: ...ray because of air bubble Flush the flow cell see page 3 4 or apply slight backpressure of 207 to 345 kPa 2 to 3 bar 30 to 50 psi on the detector waste outlet For example connect a 1 to 2 foot 30 to 6...

Page 72: ...from flow cell gasket Replace the flow cell see page 3 5 Leak from flow cell inlet and outlet fittings Check fittings for overtightening or undertightening and replace fittings if necessary PDA detect...

Page 73: ...e shapes of the absorbance spectra It also explains how spectral contrast represents those spectra as vectors determining whether differences among them arise from the presence of multiple compounds i...

Page 74: ...7 for compound B Note that this comparison of a single wavelength pair s absorbance ratios yields little information about a compound For more information you must compare the ratios of multiple wavel...

Page 75: ...tor for the resultant spectrum increases A detector derived vector can include absorbances in the range of 190 to 800 nm To enhance spectral sensitivity set the bench resolution to 1 2 nm Tip Do not i...

Page 76: ...he number of wavelengths from the spectrum To compare two spectra the spectral contrast algorithm forms a vector for each spectrum in an n dimensional space The two spectral vectors are compared mathe...

Page 77: ...hown in the next three figures Spectra with different shapes In the following figure the absorbance spectra of two compounds A and B are distinctly different They therefore produce a large spectral co...

Page 78: ...absorbance properties of different compounds For example multiple spectra of the same compound may exhibit slight differences because of detector noise photometric error high sample concentration or...

Page 79: ...centration Variation in solvent composition These sources of spectral variation can cause chemically pure baseline resolved peaks to exhibit a small level of spectral inhomogeneity You can assess the...

Page 80: ...d be less than 1 AU Keep in mind that the absorbance of the mobile phase reduces the working linear dynamic range by the amount of mobile phase absorbance at each wavelength For examples of mobile pha...

Page 81: ...o nonideal phenomena alone and that no evidence exists for significant differences between the spectra A spectral contrast angle greater than its threshold angle indicates that the shape differences a...

Page 82: ...5 10 Spectral Contrast Theory...

Page 83: ...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 84: ...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 85: ...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 86: ...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 87: ...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 88: ...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 89: ...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 90: ...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 91: ...Warnings that apply to all Waters instruments A 9 Nonmetallic Tetrahydrofuran THF Methylene chloride Dimethyl sulfoxide THF...

Page 92: ...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 93: ...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 94: ...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 95: ...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 96: ...A 14 Safety Advisories...

Page 97: ...6 kg 27 75 pounds Environmental specifications Attribute Specification Operating temperature 4 to 40 C 39 2 to 104 F Operating humidity 95 noncondensing Shipping and storage temperature 30 to 60 C 22...

Page 98: ...e their electrical power from a local level such as an electrical wall outlet c Pollution Degree 2 A measure of pollution on electrical circuits which may produce a reduction of dielectric strength or...

Page 99: ...trile 90 10 Drift UV dry 1000 AU hr Filter 1 second 30 second segments Wavelength 254 nm Bandwidth 3 6 nm 3 pixel bunch Warm up time 60 minutes Environmental stability 2 C hr Flow cell analytical 10 m...

Page 100: ...mm Tubinginternal diameter inches Pressure rating bar psi Inlet Outlet Analytical 9 3 10 0 010 0 010 69 1000 Auto purification analytical prep 12 4 0 5 0 010 0 040 0 040 138 2000 Microbore 4 1 8 0 0 0...

Page 101: ...Contents Topic Page Introduction C 2 Solvent miscibility C 3 Buffered solvents C 6 Head height C 6 Minimum tubing bend radius recommendations C 6 Solvent viscosity C 7 Mobile phase solvent degassing...

Page 102: ...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 103: ...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 104: ...17 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 Diox...

Page 105: ...e 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 end of the scale...

Page 106: ...minimum of 15 mL for sparge equipped units and a minimum of 45 mL for in line vacuum degasser equipped units Head height Position the solvent reservoirs at a level above the HPLC equipment or on top o...

Page 107: ...lysis is not known monitor the pressure during the run using the Chart Out terminal Mobile phase solvent degassing Mobile phase difficulties account for 70 or more of all liquid chromatographic proble...

Page 108: ...ou heat the solvent If the heat of solution is endothermic the solubility increases when you heat the solvent For example the solubility of He in H2O decreases with an increase in temperature but the...

Page 109: ...ent Henry s Law states that the mole fraction of a gas dissolved in liquid is proportional to the partial pressure of that gas in the vapor phase above the liquid If the partial pressure of a gas on t...

Page 110: ...rs in series The in line degasser is available as an option or factory installed in the Waters 2695 Separations Module XE model Wavelength selection This section includes UV cutoff ranges for common s...

Page 111: ...245 Methylene chloride 233 Cyclohexane 200 n Pentane 190 Cyclopentane 200 n Propanol 210 Diethyl amine 275 n Propyl chloride 225 Dioxane 215 Nitromethane 380 Ethanol 210 Petroleum ether 210 Ethyl ace...

Page 112: ...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 CHAPS...

Page 113: ...rsity possible within a given sample scanning over a range of wavelengths may be necessary to determine the best wavelength for a particular analysis Electronic absorption bands of representative chro...

Page 114: ...the chosen detection wavelengths With such a mobile phase ensure that any Nitro NO2 210 strong Nitrile ONO 220 230 1000 2000 300 400 10 Azo N N 285 400 3 25 Nitroso N O 302 100 Nitrate ONO2 270 should...

Page 115: ...the table below are based on a 10 mm pathlength Mobile phase absorbance measured against air or water Absorbance at specified wavelength nm 200 205 210 215 220 230 240 250 260 280 Solvents Acetonitril...

Page 116: ...1 2 33 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...

Page 117: ...um 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...

Page 118: ...02 0 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...

Page 119: ...ng bubbles from flow cell 4 7 connecting chart recorder 2 13 electricity source 2 5 Empower data system 2 14 Ethernet cable 2 6 external analog data collection device 2 13 inject start 2 7 injection t...

Page 120: ...alling 3 6 maintaining 3 4 removing 3 5 replacing 3 5 specifications B 4 TaperSlit 1 4 unpacking 2 2 flushing flow cell 3 4 fuses replacing 3 10 G gas requirements 2 18 solubility C 7 C 8 generating i...

Page 121: ...ity angle photometric error effects 5 8 purpose and audience iv R reading calibration constants 4 4 lamp energy 4 3 reagents C 18 rear panel interface connections changing 4 5 displaying 4 4 reference...

Page 122: ...ors 5 2 data resolving 1 5 resolution averaging 1 10 spectrum match spectral shape differences 5 7 startup procedure 2 19 stop flow generating 2 12 symbols caution A 5 electrical A 12 handling A 13 wa...

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