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(PDA) detectors

1. Warning symbols alert you to the risk of death injury or seriously adverse physiological reactions associated with an instrument s use or misuse Heed all warnings when you install repair and operate Waters instruments Waters assumes no liability for the failure of those who install repair or operate its instruments to comply with any safety precaution Task specific hazard warnings The following warning symbols alert you to risks that can arise when you operate or maintain an instrument or instrument component Such risks include burn injuries electric shocks ultraviolet radiation exposures and others When the following symbols appear in a manual s narratives or procedures their accompanying text identifies the specific risk and explains how to avoid it Warning General risk of danger When this symbol appears on an instrument consult the instrument s user documentation for important safety related information before you use the instrument AX Warning Risk of burn injury from contacting hot surfaces A Warning Risk of electric shock Ay Warning Risk of fire A Warning Risk of sharp point puncture injury A Warning Risk of hand crush injury AX Warning Risk of exposure to ultraviolet radiation AQ Warning Risk of contacting corrosive substances JAN Warning Risk of exposure to a toxic substance AAN Warning Risk of personal exposure to laser radiation A 2 Safety Advisories
2. 5 4 Acetone 0 32 56 3 15 17 330 5 5 Benzyl alcohol 5 80 205 5 13 5 7 Methoxyethanol 1 72 124 6 13 6 2 Acetonitrile 0 37 81 6 11 17 190 6 2 Acetic acid 1 26 117 9 14 6 4 Dimethylformamide 0 90 153 0 12 C 4 Solvent Considerations Solvent miscibility Continued Boilin Miscibilit pda Solvent Sec Point C Number d m i 1 atm M 6 5 Dimethylsulfoxide 2 24 1890 9 6 6 Methanol 0 60 64 7 12 210 7 3 Formamide 3 76 210 5 3 9 0 Water 1 00 100 0 How to use miscibility numbers Use miscibility numbers M numbers to predict the miscibility of a liquid with a standard solvent see page C 3 To predict the miscibility of two liquids subtract the smaller M number value from the larger M number value Ifthe difference between the two M numbers is 15 or less the two liquids are miscible in all proportions at 15 C A difference of 16 indicates a critical solution temperature from 25 C to 75 C with 50 C as the optimal temperature Ifthe difference is 17 or greater the liquids are immiscible or their critical solution temperature is above 75 C Some solvents prove immiscible with solvents at both ends of the lipophilicity 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 end of t
3. Ra ET ee EE iii Salety EE E GT EE iii re EE AE iv Operating this instrument sssssesssesseoesocoeccsseccceooeocceecoeoceecoeeoceeceeeceeeceecesecceeceee iv EEN iv Audience and purpose seno iv Intended use of the 2998 Photodiode Array detector cccccccccceccsecceseeeeeeeeeees v E E v BEE E v ISM classification egent deeg v ISM Classification ISM Group 1 Class B ercsnsniiisinosranirn ni v EC authorized representative sssssscssssssssssssssssssscssssssccssssssccscccccccsccesecesceees vi 1 2998 PDA Detector Optics Principles sssssssssssssssssscececeeeees 1 1 Detector OptiCS ssiisisswesssaccsssasssssssaiaiveceassassasdsaasedckscasdusansasssetaaasaasedeasceeaaseeceassisanes 1 2 Keren 1 3 Flow Cell Operating Principles sssesseesssessssssesssesssesssossosssosssossoossoossessoosseossoo 1 4 Resolving Spectral data cccccccccccccccccecccccccccceccecccecccecceeccececeecseecececseeseceese 1 5 Measuring light at the photodiode array esssssssssssssssssosssosssossossssossossososeoo 1 6 Optimizing the signal to noise PALO siisesieidsicieessiseidctiassincindi cheater presiecereriarers 1 7 CL iia EE 1 7 Selecting the appropriate sampling rate ccccccccccecceccceceeeeeeceseeceeceseceeeess 1 7 Computing absorbance data points cccccccccceccecccccceccceccceccscccesccsccssccsscess 1 9 LCP Ee a EE 1 9 Table of Contents vii EE Spee ra a TE 1 9 IEN 1 9 EE Eege 1 12 Reference wavelength comp
4. one low Note that the spectral contrast angle between these absorbance spectra of the same compound is 3 4 Spectral Contrast Theory Normalized absorbance spectra of a compound at two concentrations Normalized spectra of a compound at different concentrations Spectral contrast angle 3 4 Normalized absorbance Region of little or no analyte absorption 22000 240 00 26000 230 00 300 00 320 00 340 00 Wavelength nm Undesirable effects Shape differences between absorbance spectra can be caused by one or more of the following undesirable effects Detector noise Photometric error caused by high sample concentration e 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 significance of spectral inhomogeneity by comparing a spectral contrast angle to a threshold angle see page 5 8 Undesirable effects 5 7 Detector noise Statistical and thermal variations add electronic noise to the detector s absorbance measurements The noise which manifests itself as fluctuations in the baseline is known as baseline noise The magnitude of any absorbance differences caused by statistical and thermal variations can be predicted from the instrument noise in the baseline region of a chromatogram Photometric error At high absorbances generally those greater than 1 AU a combina
5. power 2 20 4 6 light path optics assembly 1 2 line spikes 4 6 M maintaining flow cell 3 4 maintenance considerations 3 2 routine 3 3 mass spectrometer shock hazard A 4 MassLynx data system connecting 2 14 match angle photometric error effects 5 8 maximum absorbance 5 8 miscibility of solvents C 3 C 5 mobile phase wavelengths C 14 monitoring detector LEDs 2 20 N network installation guidelines 2 7 noise effects 5 7 O operational specifications B 2 optics assembly light path 1 2 P photodiode array measuring light 1 6 photometric error 5 8 physical specifications B 1 plumbing connections making 2 16 power LED 2 20 4 6 power surges 4 6 purity 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 spectrum 1 9 wavelength 1 13 removing cuvette 2 24 flow cell 3 5 lamp 3 7 replacing cuvette holder 2 24 flow cell 3 5 fuses 3 10 lamp 3 7 reservoirs positioning C 6 resolution comparison 1 11 spectral 1 9 S safety advisories A 1 safety considerations maintenance 3 2 sampling rate comparison 1 8 selecting 1 7 serial number locating 2 2 shutting down 2 21 signal cables connecting 2 5 connections inject start 2 7 injection trigger 2 15 input and output 2 9 manual injector 2 8 signal to noise ratio optimizing 1 7 sol
6. publication Biosafety in Microbiological and Biomedical Laboratories BMBL Chemical hazard warning This warning applies to Waters instruments that can process corrosive toxic flammable or other types of hazardous material A A Warning Waters instruments can be used to analyze or process potentially hazardous substances To avoid injury with any of these materials familiarize yourself with the materials and their hazards observe Good Laboratory Practices GLP and consult your organization s safety representative regarding proper use and handling Guidelines are provided in the latest edition of the National Research Council s publication Prudent Practices in the Laboratory Handling and Disposal of Chemicals Caution symbol The caution symbol signifies that an instrument s use or misuse can damage the instrument or compromise a sample s integrity The following symbol and its associated statement are typical of the kind that alert you to the risk of damaging the instrument or sample CO Caution To avoid damage do not use abrasives or solvents to clean the instrument s case Caution symbol A 5 Warnings that apply to all Waters instruments A A 6 When operating this device follow standard quality control procedures and the equipment guidelines in this section Attention Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user s au
7. 1 SDS 0 1 0 02 0 01 lt 0 01 Triton 2 48 2 50 2 43 2 42 2 387 2 87 0 50 0 25 0 67 1 42 X 100 0 1 Tween 20 0 21 0 14 0 11 0 10 0 09 0 06 0 05 0 04 0 04 0 03 0 1 C 18 Solvent Considerations Index A absorbance calculating 1 3 maximum 5 8 photometric error 5 8 spectra comparing 5 2 absorbance data points computing 1 9 acids C 16 analog signals 2 8 2 13 audience and purpose iv averaging chromatographic sampling rate 1 11 spectral resolution 1 10 B Beer s law 1 4 5 8 benefits of degassing C 9 biohazard warning A 5 buffered solvents C 6 burst warning A 3 CG calibration constants reading 4 4 verifying 4 2 caution symbol A 5 chart recorder connecting 2 13 chemical hazard warning A 5 chromatographic sampling rate averaging 1 11 clearing 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 trigger signals 2 15 input and output 2 9 manual injector 2 8 MassLynx data system 2 14 signal cables 2 5 cooling time lamp 3 7 cuvette holder illustrated 2 22 removing 2 24 replacing holder 2 24 using 2 21 D dark current 1 9 data filtering 1 12 rates 1 12 degassing benefits C 9 considerations C 9 C 10 derived vectors 5 3 5 4 detector dark current 1 9 dimensions 2 4 installing 2 2 n
8. 4 Spectral contrast ANGIE S ssscessesrssessecsscessessscsssesssssscsscossosssccscocsccssccssccescoess 5 4 Undesirable effects ssis ra EE a 5 7 Detector To Necris EA 5 8 PHO Te or Of conem iad ewe ees 5 8 hl 5 8 Threshold angle ccccccccccccesccecccecceccceceseccssccceccsscesecceccsscesssesecesecesecescesscenss 5 8 A Safety AdVisOriES cisccsccssnsiasssseusssisadsasssevnisenncesseasdesbaesawersaaddadeacibuerbunedeces A 1 ET le EE A 2 Task epecilie hazard Warning S osson uS E TE TAES A 2 EE A 3 Table of Contents ix x Caution TE A 5 Warnings that apply to all Waters instruments cccccccccccsecceceeseesseeecs A 6 Electrical and handling symbols cccssssssesssssssesscssssssssssssssssssssssssssees A 12 NEE A 12 Tadine SS say sheave erent eaves Menten Oem en eas A 13 EE B 1 Solvent Considerations sseessessseesossssessssseeseeececoceeccccssssessseeeeceeeeecoseose C 1 Vx GH OCU CUNO gege eege SEENEN desea aaa aa Ea C 2 Clean GOLF EE C 2 a ER C 2 Preparation check iatasuaoucesiuni cairan ra a R A mnie C 2 KE C 2 ae Ee E C 2 Reb P A a Girls E C 3 Soly nt miscibility E C 3 How to use miscibility nan B61 occ cssicosiseeeiwneesineevian cotinine sruteiawreswereieescervovesies C 5 Buffered Solvents cesscsescasccscsdscesascussessesedewsscasescccusasssseusesessuaceseesecdsevecseseeseoreseues C 6 Head Net ht ses csessseesssdoscesesesce can tevcassseshwdesecoscasseuccsdecececutes decebestdsbeccevesceeeccs
9. Warning Risk of exposure to biological agents that can pose a serious health threat AA Warning Risk of tipping AAN Warning Risk of explosion Warning Risk of eye injury Specific warnings The following warnings can appear in the user manuals of particular instruments and on labels affixed to them or their component parts Burst warning This warning applies to Waters instruments fitted with nonmetallic tubing Warning Pressurized nonmetallic or polymer tubing can burst Observe these precautions when working 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 tetrahydrofuran THF and nitric or sulfuric acids Be aware that some compounds like methylene chloride and dimethyl sulfoxide can cause nonmetallic tubing to swell which significantly reduces the pressure at which the tubing can rupture Warning symbols A 3 Mass spectrometer flammable solvents warning This warning applies to instruments operated with flammable solvents Warning Where significant quantities of flammable solvents are involved a continuous flow of nitrogen into the ion source is required to prevent possible ignition in that enclosed space Ensure that the nitrogen supply pressure never falls below 690 kPa 6 9 bar 100 psi during an analysis in which flammable solvents are used Als
10. be attributed to nonideal phenomena Spectral Contrast Theory Comparison of a spectral contrast angle to its threshold angle can help determine whether the shape difference between spectra is significant In general a spectral contrast angle less than its threshold angle indicates that shape differences are attributable to 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 arise from significant differences between the spectra When automating the spectral contrast comparison the maximum absorbance of the spectra must not exceed 1 AU Effects of pH on the absorbance spectrum of p aminobenzoic acid Effect of pH Absorbance Wavelength nm Undesirable effects 5 9 5 10 Spectral Contrast Theory Safety Advisories Waters instruments display hazard symbols designed to alert you to the hidden dangers of operating and maintaining the instruments Their corresponding user guides also include the hazard symbols with accompanying text statements describing the hazards and telling you how to avoid them This appendix presents all the safety symbols and statements that apply to the entire line of Waters products Contents Topic Page Warning symbols A 2 Caution symbol A 5 Warnings that apply to all Waters instruments A 6 Electrical and handling symbols A 12 A 1 Warning symbols
11. can use the console to determine the status of input output signal connections 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 connected to the terminal A red LED symbol indicates no signal cable is connected to the terminal To display 2998 PDA detector rear panel interface connections 1 Inthe console select 2998 PDA Detector from the system tree 2 Inthe PDA detector information window click Troubleshoot gt Rear panel The PDA Detector Rear Panel dialog box appears 4 4 Diagnostic Tests and Troubleshooting The following table describes the PDA detector I O connections PDA detector analog out event in connections Signal connections Analog 1 Out Description Method programmable analog output Analog 2 Out Method programmable analog output Switch 1 Out Controls a timed event or threshold level and is a user programmable auxiliary output Switch 2 Out Controls a timed event or threshold level and is a user programmable auxiliary output Inject Start In Activates timed events by triggering the run time clock to start Lamp Off In When the input is triggered the lamp is turned off Chart Mark In 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 con
12. click OK Replacing the fuses AN Warning To avoid electric shock power off and unplug the 2998 PDA detector before examining the fuses For continued protection against fire replace fuses with those of the same type and rating only The detector requires two 100 to 240 VAC 50 to 60 Hz F 3 15 A 250 V fast blow 5 X 20 mm IEC fuses Suspect a fuse is open or otherwise defective when the detector fails to power on the fan does not operate To replace the fuses Requirement Replace both fuses even when only one is open or otherwise defective 1 Power off the detector and disconnect the power cord from the power entry module 2 Pinch the sides of the spring loaded fuse holder located above the power entry module on the rear panel of the detector 3 With minimum pressure withdraw the spring loaded fuse holder 3 10 Maintaining the Detector Removing the fuseholder Fuses Power entry module Fuse holder Remove and discard the fuses 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 until the assembly locks into position Reconnect the power cord to the power entry module Replacing the fuses 3 11 3 12 Maintaining the Detector Diagnostic Tests and Troubleshooting Consult this chapter when troubleshooting problems with the 2998 PDA detector However bear
13. front plate Grasp the handle and gently pull it toward you 6 Unpack and inspect the new flow cell 7 Square the flow cell assembly in front of the opening and then insert it into the optics bench 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 Alignment pin Flow cell handle 9 Continue to insert the flow cell until the three thumbscrews align with their holes in the bulkhead Oo Caution To prevent the flow cell from binding and ensure that it is properly seated in the bulkhead alternate between tightening the captive screws and pushing the flow cell forward 3 6 Maintaining the Detector 10 11 12 13 Hand tighten the thumbscrews and then tighten them snug with the 1 4 inch flat blade screwdriver Connect the inlet tubing to the column outlet connection and flow cell inlet Replace the front panel cover Before you power on the detector prime the system to fill the flow cell with solvent and remove any air Replacing the lamp Change the lamp when it repeatedly fails to ignite or when the detector fails to calibrate The 2998 detector source lamp is warranted to light and pass startup diagnostic tests for 2000 hours or 1 year from the date of purchase whichever comes first Warning To prevent burn injuries allow the lamp to cool for
14. 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 chromatography or other system instruments Contents Topic Page Diagnostic tests 4 2 General troubleshooting 4 6 4 1 Diagnostic tests The 2998 PDA detector automatically runs a series of internal diagnostic tests upon start up The indicator LEDs on the front of the detector and messages at the Empower workstation show the results of the start up internal diagnostic tests If you need to determine the cause of a problem during operation of the detector you can run the same internal diagnostic tests from the Empower workstation Additional information about the performance of the detector is also available through the PDA Calibration window accessed from Run Samples in the Empower software Verifying detector calibration 4 2 Verify the PDA detector calibration after removing and replacing a flow cell Before you power on the detector prime the system to fill the 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 Caution To ensure the detector is properly aligned and calibrated the flow cell must be filled with solvent before you power on the detector An empty flow cell will cause a calibration error To verify detecto
15. number on the rear panel nameplate or inside the front panel corresponds to the number on the instrument validation certificate Setting Up the Detector Laboratory site selection To ensure the reliable operation of your detector e do not situate it under a heating or cooling vent connect it to a power supply that is grounded AC 100 to 240 VAC provide clearance of at least 15 cm 6 inches on the back side for ventilation a Caution To avoid damaging the detector the weight of items stacked on top of it should not exceed 18 1 kg 40 pounds The dimensions shown in the figure Detector dimensions 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 resolution of the chromatogram Requirement Mount the detector on a level surface to allow proper function of the drip management system drain tube to which you can connect a waste reservoir that diverts solvent leaks from the flow cell Laboratory site selection 2 3 Detector dimensions The following figure shows the dimensions of the detector Detector dimensions 61cm eger 24 inches a 19 1 cm 7 64 inches 13 5 inches nN Connecting to the electricity source The 2998 PDA detector requires a separate grounded electricity source The ground connection in the electrical outlet must b
16. of the detector to the Off O position Using a cuvette The detector cuvette option allows for ease of use in Sample handling Instrument verification and qualification 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 detector flow cell assembly Using a cuvette 2 21 2998 PDA Detector cuvette holder with the cuvette inserted Frosted sides of cuvette facing up and down Restriction Because the measurement is actually a composite of both the contents of the cuvette and the flow cell you need to perform cuvette measurements under identical flow cell conditions If you store spectra and acquire new spectra for subtraction you need to be aware of differences if any in flow cell conditions Ideally you should perform both the zero and sample measurements using the cuvette when the HPLC instruments are in the idle or static state and operating under identical flow cell conditions Caution Handle the cuvette gently and on the frosted sides only Fingerprints on the clear quartz interfere with the light path and compromise the integrity of cuvette measuring operations Before you begin Recommendation To ensure accurate results use a 10 mm path length quartz cuvette and matched pairs from the same manufacturing lot of quartz cuvettes for your zero and sample measu
17. panel cover e To prevent invalid subsequent chromatographic results remove the cuvette from the detector and replace the empty holder after running your cuvette measurements To maintain 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 measurement 7 Replace the reference cuvette with a cuvette containing your analyte dissolved in mobile phase solvent and run a sample measurement 8 Use the storage review subtract and review and replay functions to analyze the data obtained 2 24 Setting Up the Detector Maintaining the Detector This chapter describes the 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 flow cell 3 4 Replacing the lamp 3 7 Replacing the fuses 3 10 3 1 Contacting Waters technical service If you are located in the USA or Canada report malfunctions or other problems to Waters Technical Service 800 252 4752 Otherwise phone the Waters corporate headquarters in Milford Massachusetts USA or contact your local Waters subsidiary Our Web site includes phone numbers and e mail addresses for Waters locations worldwide Go to www waters com and click About Waters gt Worldwide Offices When
18. replace fittings if necessary 4 10 Diagnostic Tests and Troubleshooting Spectral Contrast Theory The spectral contrast algorithm compares the UV Vis absorbance spectra of samples the detector collects This chapter describes the theory on which the algorithm is based explaining how it exploits differences in the 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 in the same peaks coelution or from nonideal conditions like noise photometric error or solvent effects Contents Topic Page Comparing absorbance spectra 5 2 Representing spectra as vectors 5 2 Spectral contrast angles 5 4 Undesirable effects 5 7 5 1 Comparing absorbance spectra When measured at specific solvent and pH conditions the shape of a compound s absorbance spectrum characterizes the compound The varying extent of UV Vis absorbance occurring at different wavelengths produces a unique spectral shape The following figure shows the absorbance spectra for two compounds A and B The ratio of the absorbance at 245 nm to that at 257 nm is about 2 2 for compound A and 0 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 wavelength pairs Comparing s
19. shall be made aware that if the equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impaired Attention L utilisateur doit tre inform que si le mat riel est utilis d une fa on non sp cifi e par le fabricant la protection assur e par le mat riel risque d tre d fectueuses Vorsicht Der Benutzer wird darauf aufmerksam gemacht dass bei unsachgem er Verwenddung des Ger tes die eingebauten Sicherheitseinrichtungen unter Umst nden nicht ordnungsgem funktionieren Attenzione si rende noto all utente che l eventuale utilizzo dell apparecchiatura secondo modalit non previste dal produttore pu compromettere la protezione offerta dall apparecchiatura Advertencia el usuario deber saber que si el equipo se utiliza de forma distinta a la especificada por el fabricante las medidas de protecci n del equipo podr an ser insuficientes zs EF a EE a Ta AE mm R i A a R RERA EI AEH SE Ri A THE BE a TS BCE 3B BR HAAMER A AER te TI E TE EA ABA THe BE ICR URE CH 35 o AD MASAMI SACHA BS HACe AUS ASS SS Sul Hot PS SHO MOS SSoA FS UE SS NSAAAl DCH IA AHO SLICE BG 2 Pld ROEI E V RE SN CWRW AE Chea EAT SL eae Hep LCV StRaEDS EMIT 72D net A OO LICE UT FAV A 10 Safety Advisories Warning To protect against fire replace fuses with t
20. tubing under pressure Always wear eye protection when near pressurized polymer tubing Extinguish all nearby flames Do not use tubing that has been severely stressed or kinked Do not use nonmetallic tubing with tetrahydrofuran THF or concentrated nitric or sulfuric acids Be aware that methylene chloride and dimethyl sulfoxide cause nonmetallic tubing to swell which greatly reduces the rupture pressure of the tubing Attention Manipulez les tubes en polym re sous pression avec precaution Portez syst matiquement des lunettes de protection lorsque vous vous trouvez a proximit de tubes en polym re pressuris s Eteignez toute flamme se trouvant proximit de l instrument Evitez d utiliser des tubes s v rement d form s ou endommag s Evitez d utiliser des tubes non m talliques avec du t trahydrofurane THF ou de l acide sulfurique ou nitrique concentr Sachez que le chlorure de m thyl ne et le dim thylesulfoxyde entra nent le gonflement des tuyaux non m talliques ce qui r duit consid rablement leur pression de rupture Vorsicht Bei der Arbeit mit Polymerschl uchen unter Druck ist besondere Vorsicht angebracht In der N he von unter Druck stehenden Polymerschl uchen stets Schutzbrille tragen Alle offenen Flammen in der N he l schen Keine Schl uche verwenden die stark geknickt oder berbeansprucht sind Nichtmetallische Schl uche nicht f r Tetrahydrofuran THF oder konzentrierte Salp
21. within a range of 190 to 800 nm The figure below illustrates the light path through the optics assembly of the detector Optics assembly light path Grating Order filter Photodiode Window Is array M1 mirror mirror and mask Lamp Filter Flow cell flag shutter The following table describes the optics assembly components Optics assembly components Component Function Lamp Deuterium source lamp M1 mirror Focuses light from the deuterium source lamp 1 2 2998 PDA Detector Optics Principles Optics assembly components Continued Component Function Window Used to help minimize air infiltration into the lamp housing Filter flag shutter Flag positions for measuring open sample and blocked dark beam energies and a third for wavelength verification Flow cell Houses the segment of the flow path containing eluent and sample through which the polychromatic light beam passes Spectrograph The mirror focuses light transmitted through the flow mirror and mask cell onto the slit at the entrance to the spectrographic portion of the optics The mirror mask defines the size of the beam at the grating Slit Determines wavelength resolution and intensity of light striking the photodiodes The width of the slit is 50 um Grating Disperses light into bands of wavelengths and focuses them onto the plane of the photodiode array Order filt
22. you contact Waters be prepared to provide this information Nature 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 For complete information on reporting shipping damages and submitting claims see Waters Licenses Warranties and Support Services Maintenance considerations Safety and handling Observe these warning and caution advisories when you perform maintenance on your detector Warning To prevent injury always observe Good Laboratory Practices when you handle solvents change tubing or operate the system Know the physical and chemical properties of the solvents you use See the Material Safety Data Sheets for the solvents in use 3 2 Maintaining the Detector A Waring Avoid electric shock Do not open the detector cover The detector does not contain user serviceable components Power off and unplug the detector before performing any maintenance on the instrument D Caution To avoid damaging electrical parts never disconnect an electrical assembly while power is applied to the detector To interrupt power to the detector set the power switch to Off and then unplug the power cord from the AC outlet Wait 10 seconds before you disconnect an assembly Spare parts Waters recommends that you replace only parts mentioned in this document Fo
23. 0 69 1000 Auto purification analytical 12 4 0 5 0 010 0 040 1388 2000 prep 0 040 Microbore 4 1 8 0 0 005 0 005 69 1000 Semi preparative 18 3 3 0 0 020 0 020 69 1000 Cuvette N A 10 0 N A N A N A B 4 Specifications Solvent Considerations This appendix contains certain solvent considerations you must take into account when operating or maintaining the 2998 PDA Detector 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 C 7 Solvent degassing methods C 8 Wavelength selection C 10 Warning To avoid chemical hazards always observe Good Laboratory Practices when operating your system C 1 Introduction Clean solvents Clean solvents provide reproducible results and permit you to operate with minimal instrument maintenance A dirty solvent can cause baseline noise and drift It can also block the solvent filters with particulate matter Solvent quality Use HPLC grade solvents for the best possible results Filter solvents through 0 45 um filters before their use Solvents distilled in glass generally 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 good resolution Filter solvents
24. 20 samples second 25 msec exposure 2 exposures sample Computing absorbance data points 1 11 The readings are averaged and reported as a single data point The baseline noise decreases as the number of exposures sample increases Tip The amount of data stored is based on wavelength range spectral resolution runtime and sample rate Specify these parameter values in the General tab of the PDA Instrument Method Editor For details refer to the Empower or MassLynx online Help Filtering data In the General tab of the PDA Instrument Method Editor for details refer to the Empower or MassLynx 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 Digital filter settings for data rates Data Slow Normal Fast Increment Rate Range sec sec sec sec sec Hz 1 4 000 2 000 1 000 No filter 0 100 to 0 100 5 000 2 2 000 1 000 0 500 No filter 0 100 to 0 100 5 000 5 0 800 0 400 0 200 No filter 0 100 to 0 100 2 000 10 0 400 0 200 0 100 No filter 0 100 to 0 100 1 000 20 0 200 0 100 0 050 No filter 0 050 to 0 050 0 500 40 0 100 0 050 0 025 No filter 0 025 to 0 025 0 250 80 0 050 0 025 0 0125 No filter 0 0125 to 0 0125 0 100 Tip For best chromatography resolution choose Fast to avoid peak broadening 1 12 2
25. 280 Potassium phosphate dibasic K HPO 10 mM 0 53 0 16 0 05 0 01 lt 0 01 Sodium acetate 10 mM 1 85 0 96 0 52 0 30 0 03 lt 0 01 Sodium chloride 1M 2 00 1 67 0 40 0 10 lt 0 01 Sodium 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 lt 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 lt 0 01 Tris HC 20 mM pH 7 0 1 40 0 77 0 28 0 10 0 04 lt 0 01 Tris HCl 20 mM pH 8 0 1 80 1 90 0 43 0 13 lt 0 01 Wavelength selection C 17 Mobile phase absorbance measured against air or water Continued Absorbance at specified wavelength nm 200 205 210 215 220 230 240 250 260 280 Waters PIC reagents PIC A 0 67 0 29 0 13 0 05 0 03 0 02 0 02 0 02 0 02 lt 0 01 1 vial L PIC B6 1 2 46 2 50 2 42 2 25 1 83 0 638 0 07 lt 0 01 vial L PIC B6 low 0 01 lt 0 01 UV 1 vial L PIC D4 1 0 03 10 03 10 03 10 03 10 02 10 02 0 02 0 02 0 02 0 01 vial L Detergents BRIJ 35 1 0 06 10 03 10 02 10 02 0 02 0 01 lt 0 01 CHAPS 2 40 2 32 1 48 10 80 10 40 0 08 10 04 10 02 0 02 0 01 0
26. 2998 Photodiode Array Detector Operator s Guide 71500121902 Revision C Waters THE SCIENCE OF WHAT S POSSIBLE Copyright Waters Corporation 2010 All rights reserved Copyright notice 2010 WATERS CORPORATION PRINTED IN THE UNITED STATES OF AMERICA AND IN IRELAND ALL RIGHTS RESERVED THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER The information in this document is subject to change without notice and should not be construed as a commitment by Waters Corporation Waters Corporation assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Waters Corporation be liable for incidental or consequential damages in connection with or arising from its use Trademarks Waters and Alliance are registered trademarks of Waters Corporation and Empower MassLynx SAT IN TaperSlit and THE SCIENCE OF WHAT S POSSIBLE are trademarks of Waters Corporation Tygon is a registered trademark of Saint Gobain Ceramics amp Plastics Inc Other registered trademarks or trademarks are the sole property of their owners Customer comments Waters Technical Communications department invites you to tell us of any errors you encounter in this document or to suggest ideas for otherwise improving it Please help us better understand wha
27. 30 minutes before removing it The lamp housing gets extremely hot during operation Warning To avoid eye injury from ultraviolet radiation exposure power off the detector before changing the lamp wear eye protection that filters ultraviolet light keep the lamp in the housing during operation To remove the lamp 1 2 Power off the lamp Power off the detector and disconnect the power cable from the rear panel Allow the lamp to cool for 30 minutes Warning The lamp and lamp housing can be hot Wait 30 minutes after powering off the detector for these components to cool before touching them Lift up the front panel cover and pull it away from the chassis Replacing the lamp 3 7 5 Detach the lamp power connector from the detector Removing the lamp Alignment pin Captive screw Lamp base connector Warning Lamp gas is under slight negative 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 dispose of it 6 Loosen the two captive screws in the lamp base Gently withdraw the lamp from the lamp housing To install the lamp Caution Do not touch the glass bulb of the new lamp Dirt or fingerprints adversely affect detector operation If the bulb needs cleaning gently rub it with ethano
28. 5 gt 2 5 2 5 1 45 tetrahydro furan THF old Wavelength selection C 15 Mobile phase absorbance measured against air or water Continued Absorbance at specified wavelength nm 200 205 210 215 220 230 240 250 260 280 Acids and bases Acetic acid 2 61 2 63 2 61 2 43 2 17 0 87 0 14 0 01 lt 0 01 1 Hydro 0 11 0 02 lt 0 01 chloric acid 0 1 Phosphoric lt 0 01 acid 0 1 Trifluoro 1 20 0 78 0 54 10 34 0 22 0 06 lt 0 02 lt 0 01 acetic acid Diammo 1 85 0 67 0 15 10 02 lt 0 01 nium phosphate 50 mM Triethy 2 83 2 42 2 50 2 45 2 87 1 96 0 50 0 12 0 04 lt 0 01 lamine 1 Buffers and Salts Ammonium 1 88 0 94 0 53 0 29 0 15 0 02 lt 0 01 acetate 10 mM Ammonium 0 41 0 10 0 01 lt 0 01 bicarbonate 10 mM EDTA 0 11 0 07 10 06 0 04 0 03 10 03 0 02 0 02 0 02 0 02 disodium 1mM HEPES 2 45 2 50 2 87 2 08 1 50 0 29 0 03 lt 0 01 10 mM pH 7 6 MES 2 42 2 88 1 89 0 90 10 45 0 06 lt 0 01 10 mM pH 6 0 C 16 Solvent Considerations Mobile phase absorbance measured against air or water Continued Potassium phosphate monobasic KH PO 10 mM Absorbance at specified wavelength nm 200 0 03 205 lt 0 01 210 215 220 230 240 250 260
29. 998 PDA Detector Optics Principles Reference wavelength compensation A compensated reference wavelength collects wide band absorbance data in a region of the spectra where no known analytes exist It is used to reduce detector drift and wander which can affect integration quality The detector calculates the compensation value by averaging the absorbance values within a range of wavelengths that you select It then subtracts that value from the absorbance value Abs Comp t Abs t CRef where Abs Comp absorbance compensated Abs absorbance CRef compensation reference t elapsed time after start of injection A starting and ending wavelength defines the compensation reference CRef bandwidth must be gt 40 nm and lt 100 nm and fall within a range of 190 to 800 nm Tip Select a compensation reference range where no analytes are expected to appear Since responses are subtracted from the absorbance value any responses within the compensation reference range can erroneously affect quantitative data Computing absorbance data points 1 13 1 14 2998 PDA Detector Optics Principles Setting Up the Detector The chapter provides the information necessary to set 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 detector 2 16 Starting up and shutting down the detect
30. Continued Solvent aa Solvent ae Amy alcohol 210 Isopropyl ether 220 Amy chloride 225 Methanol 205 Benzene 280 Methyl acetate 260 Carbon disulfide 380 Methyl ethyl ketone 330 Carbon tetrachloride 265 Methyl isobutyl 334 ketone Chloroform 245 Methylene chloride 233 Cyclohexane 200 n Pentane 190 Cyclopentane 200 n Propanol 210 Diethyl amine 275 n Propyl chloride 225 Dioxane 215 Nitromethane 380 Ethanol 210 Petroleum ether 210 Ethyl acetate 256 Pyridine 330 Ethyl ether 220 Tetrahydrofuran 230 Ethyl sulfide 290 Toluene 285 Ethylene dichloride 230 Xylene 290 Wavelength selection C 11 Mixed mobile phases The table below contains approximate wavelength cutoffs for some other solvents buffers detergents and mobile phases The solvent concentrations represented are those most commonly used If you want to use a different concentration you can determine approximate absorbance using Beer s Law because absorbance is proportional to concentration Wavelength cutoffs for different mobile phases UV UV Mobile Phase Cutoff Mobile Phase Cutoff nm nm Acetic acid 1 230 Sodium chloride 1 M 207 Ammonium acetate 205 Sodium citrate 10 mM 225 10 mM Ammonium bicarbonate 190 Sodium dodecyl sulfate 190 10 mM BRIJ 35 0 1 190 Sodium formate 10 mM 200 CHAPS 0 1 215 Triethyl amine 1 235 Diammonium phosphate 205 Trifluoracet
31. bances that can affect the performance of this instrument do not use cables longer than 3 meters 9 8 feet when you make connections to the I O connectors In addition ensure 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 panel 2998 rear panel analog out event in connectors Connector B outputs Connector A inputs 1 Analog 1 1 Inject Start 2 Analog 1 2 Inject Start A 3 Ground 3 Ground A 4 Analog 2 4 Lamp Off A 5 Analog 2 5 Lamp Off A 6 Switch 1 6 Chart Mark 7 Switch 1 7 Chart Mark A 8 Ground 8 Ground A 9 Switch 2 9 Auto Zero Z 10 Switch 2 10 Auto Zero Making signal connections 2 9 The following table describes the detector I O connections Detector analog out event in connections Signal connections Inject start Description Activates timed events by triggering the run time clock to start Lamp off When the input is enabled the lamps is turned off The lamp can only be turned on by sending a new method 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 configurable Auto zero Calculates an offset value that when ad
32. by the diode exposure time Optimizing the signal to noise ratio To optimize signal to noise ratios choose an acquisition wavelength range that includes only the wavelengths of interest Ensure that the range is one in which the mobile phase absorbs 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 See page 1 10 for more information Optimizing filter constants The filtering constant you select affects the peak intensity To increase sensitivity decrease the filter time constant Selecting the appropriate sampling rate A sufficient number of points must fall across a peak to define its shape Thus the definition between peaks is lost at very low sampling rates Empower uses Measuring light at the photodiode array 1 7 the end time minus the start time to calculate the Points Across Peak value for each integrated peak in the chromatogram Tip The Points Across Peak value appears in the Peaks table at the bottom of the Review Main window If the Points Across Peak field is not visible right click 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 Peak value for the narrowest peak of interest is less than 25 you should specify a hig
33. cipated to be used for subsequent analysis Detector troubleshooting The following table contains general hardware troubleshooting for the detector PDA detector troubleshooting Symptom Both LEDs unlit Possible cause Corrective action No power 1 Inspect line cord connections 2 Test electrical outlet for power Open spent or Replace fuse see defective fuse page 38 10 General troubleshooting 4 7 PDA detector troubleshooting Continued Symptom Change in reference spectrum Possible cause Mobile phase contains gas or is contaminated Corrective action Prepare fresh mobile phase and degas thoroughly Air bubbles trapped in flow cell Reseat and check alignment of flow cell 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 60 cm length 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 disconnected cable Inspect cable connections tighten connectors or replace cable Configuration problem Check Ethernet configuration For details see Empower online Help Lamp light flashes green and power light constant green The detector is ini
34. connector B Analog 1 Analog 1 Ground Analog 2 Analog 2 Switch 1 Switch 1 Ground Switch 2 0 Switch 2 A OO JO OP Gah A 1 Setting Up the Detector Connecting injection trigger signals The detector accepts the following injection trigger signals from a manual injector Inject start signal from a contact closure signal with each injection e Auto zero signal to adjust the zero offset of the detector each time the injector makes an injection Each time the detector receives a signal from an injector it performs the corresponding auto zero or 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 on inject Inject start connections to an injector pulse duration 0 to 10 seconds 2998 PDA detector connector A Injector connector Pin 1 inject start red Two spade lug or similar terminal Pin 2 inject start black connectors Inject start connections to an injector 2998 PDA detector connector A Injector Inject Start Inject Start Ground Lamp Off Lamp Off Chart Mark Chart Mark Ground Auto Zero Auto Zero A OO JO Om P Go A t oO Making signal connections 2 15 Plumbing the detector See also Minimum tubing bend radius recom
35. ctor To start up the detector 1 In your instrument method set the solvent delivery system or pump to deliver 10 mL of HPLC grade water For details refer to the Empower or MassLynx online Help Guidelines Use only thoroughly degassed HPLC grade solvents Gas in the mobile phase may form bubbles in the flow cell and cause the detector to fail the reference energy 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 and ensure the volumes of solvents are sufficient for priming Press the Off On 0 1 switch on the front panel of the detector to the On 1 position Observe the Lamp and Power indicator LEDs on the front panel of the detector The power and lamp LEDs change as follows The power LED shows green During initialization the power LED flashes green After the detector has successfully powered on the power and lamp LEDs show steady green Starting up and shutting down the detector 2 19 4 Wait 1 hour for the detector to stabilize before acquiring data If the detector fails to stabilize see Chapter 4 Detector indicator lights Waters 2998 Photodiode Array Detector LAMP POWER Lamp Power indicator indicator Monitoring detector LEDs Light emitting diodes on the detector indicate its state of funct
36. ctor depend on the signal connections available on the other instruments in your HPLC system Connecting the Ethernet cable 2 6 A Waters instrument communicates with the acquisition computer through the dedicated local area network LAN At the acquisition computer the instrument network card provides the interface that makes communication possible You must install the Waters instrument software driver in the acquisition computer so that the computer can control the instrument See the software installation instructions that accompany the instrument control software Single Waters instrument connection 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 Instrument LAN network card _ Waters III instrument Acquisition computer Ethernet cable Multiple Waters instrument connections System configurations with many Waters Ethernet instruments require an Ethernet switch to communicate multiple signals among Waters instruments and the acquisition computer Connection hardware requires one standard Ethernet cable per Waters instrument and a standard Ethernet cable to connect the network switch and the acquisition computer You must install the Waters instrument control software in the acquisition computer so that the computer can control the Waters instrument Se
37. ded to the sample signal makes the resulting baseline signal zero Analog 1 and Analog 2 Method programmable analog output Minimum output voltage range 0 1 to 2 1 VDC For sample rates of 10 20 40 or 80 Hz this output runs at the selected data rate For sample rates of 1 2 or 5 Hz this output runs at 10 Hz Switch 1 Controls a timed event or threshold level and is a user programmable auxiliary output Switch 2 Controls a timed event or threshold level and is a user programmable auxiliary output Generating inject start To generate the inject start function on the detector at the start of an injection from the Alliance Separations Module make the connections shown in the following table and figure 2 10 Setting Up the Detector Tip The firmware defaults to auto zero on inject Detector connections to an Alliance separations module Alliance separations module connector B Pin 1 inject start red 2998 PDA detector connector A Pin 1 inject start red Pin 2 inject start black Pin 2 inject start black Inject start connections between the Alliance separations module and the detector Waters Alliance connector B Inject Start Inject Start Ground Stop Flow Stop Flow Hold Inject 1 Hold Inject 1 Hold Inject 2 Hold Inject 2 Ground Chart Out Chart Out E ds WE OO JO om ob A MANN 2998 PDA detector connector A I
38. e common and connected near the system N Warning Avoid electrical shock Use power cord SVT type in the United States and HAR type or better in Europe For other countries contact your local Waters distributor Power off and unplug the detector before performing any maintenance on the instrument Connect the detector to a common ground 2 4 Setting Up the Detector To connect to the electricity source Recommendation Use a line conditioner or an uninterruptible power supply UPS for optimum long term input voltage stability 1 Place the Off On 0 1 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 suitable wall outlet Important Do not turn the 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 detector with external devices 2998 PDA Detector rear panel External device inputs and outputs ok O SE o O 0 0 0 O O O O O O O O O O O O O ch O O DOS a O O 0000000000 H H OOOO OO OO OO O 000000000 Se O oo0oo000000 oo0oo000000 oo0oo000000 ESCH O H oo00 0 ve Hz VA 185 5 F 3 15A 250V Making signal connections 2 5 The signal connections you need to make to your dete
39. e the software installation instructions that accompany the software instrument driver disk Setting Up the Detector Network installation guidelines Configurations for multiple Waters instruments use a dedicated LAN which requires a design based on the following guidelines Ethernet cable A maximum distance of 100 meters 328 feet Requirement You must use a network switch for multiple Ethernet instruments Network hubs are not supported Waters Ethernet instrument connections Ethernet cable Network switch Instrument LAN IEEE card network card SE Ooo w NAAN WII IEEE 48 cable Chromatography manager Inject start cable TT Ethernet cable Chromatographic Mass PDA detector system spectrometer Making inject start signal connections When you are using an Ethernet data system with the detector the data system or controller must receive an inject start signal from the autosampler or manual injector to initiate the data collection and time based programs Making signal connections 2 7 The following table summarizes the inject start connections for different system configurations Detector inject start connections Inject start input connection on the REESEN 2998 PDA detector connector A Waters Alliance Separations Module Inject Start Waters 717 Autosampler Inject Start Waters ma
40. ectral contrast algorithm uses vectors to characterize spectra To understand the vector principle consider two vectors in the figure below which are based on the spectra depicted in the previous figure Plotting vectors for two spectra Compound B Compound A AU at 257 nm 0 0 1 0 2 0 3 0 4 AU at 245 nm In this figure the axes reflect the absorbance units of the two wavelengths used to calculate the absorbance ratio of the previous figure The head of the vector for Compound A lies at the intersection of the absorbance values for Representing spectra as vectors 5 3 Compound A at the two wavelengths represented by each axis The remaining vector is similarly derived from the spectrum of Compound B Compound B s vector points in a direction different from Compound A s Expressed by the spectral contrast angle this difference reflects the difference between the two compounds absorbance ratios at wavelengths 245 nm and 257 nm A spectral contrast angle greater than zero indicates a shape difference between spectra see Spectral contrast angles on page 5 4 Finally note that the length of the vectors is proportional to concentration Vectors derived from multiple wavelengths When absorbance ratios are limited to two wavelengths the chance that two different spectra share the same absorbance ratio is greater than if comparison is made using absorbance ratios at many wavelengths Therefore the spectral c
41. ensation 1 13 2 Setting Up the TT 2 1 Before YOU Decin sasas ea a a aa aeaa aaan 2 2 Unpacking and ins pO Ctiin 6 sccccsescssesisssssssaiessessasovsncssesscsasnsseuassosenscsesccsecusseeses 2 2 Laboratory sit selection zerek geg Ee des cascecassseses evade Sevestecnesewseoseeess 2 3 Detector dimensione oc steeds erence E 2 4 Connecting to the electricity Source ccccssscessessesssssseesscssssssesssesssssssssees 2 4 Making signal connections ccccccssecsssesssosssesssossssssosececscossccsscossccsescssosecsoess 2 5 Connecting the Ethernet cable cccccccccccccseccscecsecceececeececceececescsseesevens 2 6 Network inelallation Sieh ines sssnnnenanm onina a 2 7 Connecting to other EELER sasian aa 2 8 Plumbing the Gete Ctr ssc iiisesccieccias assisedsicadsaisdessseddanseetaccciassedassessaseesieaedeaasaass 2 16 Making the gas supply Connection asarana A 2 18 Starting up and shutting down the detector cccccccccccccccececeecsecseceess 2 18 Siariine up the E 2 19 Monitoring detector LEDS enerne anina a 2 20 chating down the delectat osia a 2 21 Using E EE 2 21 3 Maintaining the Detector sissciesdiaieicssssssiscvevsvarsveivacsitvessievedeadstevsvevesevevss 3 1 Contacting Waters technical service see EE ENEE 3 2 Maintenance considerations siceccscasacedonssnsereccasisrasscensectcensanagunsnasiceadenceassnanierens 3 2 e 3 2 ER 3 3 Routine EE EE 3 3 Maintaining the flow Cell siscccscccsesseverscacsscecsssesetecestcesasscanstee
42. er Reduces the contribution of second order diffraction of UV light less than 370 nm to the light intensity observed at visible wavelengths greater than 370 Photodiode array A linear array of 512 photodiodes The diode width 50 um together with a 50 um slit yields single wavelength resolution of 1 2 nm Calculating absorbance The detector computes absorbance by subtracting the dark current see Dark current on page 1 9 from the reference spectrum reference energy and the acquired spectrum sample energy Absorbance is based on the principles of Beer s law Beer s law The Beer Lambert law commonly called Beer s law describes the relationship between the quantity of light of a particular wavelength arriving Detector optics 1 3 at the photodiode and the concentration of the sample passing through the flow cell Beer s law is expressed as A sle where A dimensionless quantity measured in absorbance units g constant of proportionality known as the molar absorptivity l path length in centimeters 1 0 cm in the detector s normal flow cell c concentration in moles per liter Beer 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 detector element As concentration increases the chemical and instrumental requirements of Beer s law are so
43. ersely affect detector operations Be sure that the electrical supply used for the detector is properly grounded and free from any of these conditions Power LED The power LED on the detector s front panel indicates when the instrument is powered on or powered off It is constant green when the instrument is working properly Lamp LED The lamp LED to the left of the power LED indicates the lamp status Lamp LED indications LED mode and color Description Unlit Indicates the detector lamp is extinguished Constant green Indicates the detector lamp is ignited and the detector has passed power up wavelength verification Flashing green Indicates the detector is initializing or verifying wavelength calibration 4 6 Diagnostic Tests and Troubleshooting Lamp LED indications Continued LED mode and color Flashing red Description Indicates an error stopped the 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 further operation Power off the detector and then power on If the LED is still steady red contact your Waters service representative Clearing bubbles from the flow cell To clear bubbles from the flow cell Establish liquid flow through the detector flow cell with degassed acetonitrile or methanol at a flow rate that is anti
44. ess steel tubing Tubing size OD Minimum bend radius 1 16 inch or smaller 1 4 inch tubing A Solvent Considerations Minimum bend radius for stainless steel tubing Continued Tubing size OD Minimum bend radius 1 8 inch tubing 1 2 inch SET Solvent viscosity 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 mixed in different proportions can result in pressure changes during the run For example a 1 1 mixture of water and methanol produces twice the pressure of either water or methanol alone If the extent to which the pressure changes will affect the analysis is not known monitor the pressure during the run using the Chart Out terminal Mobile phase solvent degassing Mobile phase difficulties account for 70 or more of all liquid chromatographic problems Using degassed solvents is important especially at wavelengths below 220 nm Degassing provides e stable baselines and enhanced sensitivity reproducible retention times for eluting peaks reproducible injection volumes for quantitation stable pump operation Gas solubility Only a finite amount of gas can be dissolved in a given volume of liquid This amount depends on Solvent viscosity C 7 the chemical affinity of the gas for the liquid the temperature of the liqu
45. essedesss C 6 Minimum tubing bend radius recommendations ssssssssosssossoosssosssossosesso C 6 Solvent VISCOSIGY suisa e a a a aaa C 7 Mobile phase solvent degassing essesssesssesssesssessesssesssssssessssossosssesssossoessoeese C 7 BE E E C 7 Solvent degassing methods ssssssssesssesssessecossesosecsesssocoecoseccoeccescceeccesceeeseesee C 8 Eeer C 9 Ke C 9 Solvent degassing considerations cccccccccccceceseceecececccccecccceceecceseececeeseeseeens C 9 Wavelength selection scsscsccscsssescssssecevsceseaseseescecesecusestsavesesecavecsesesseeseseuseseasetes C 10 UY cutoffs tor common Sol VBI BS i isdecaataasncesvunvnvnarvduurdasunvorsnvdonedespexrdeunnvan s C 10 Ieee als E C 12 Table of Contents Wavelength selection for chromophore detect on C 13 Mobile phase 2 bs6F ane et C 14 E E Index 1 Table of Contents xi xii Table of Contents 1 2998 PDA Detector Optics Principles To use the 2998 PDA detector effectively you 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 1 5 Measuring light at the photodiode array 1 6 Computing absorbance data points 1 9 1 1 Detector optics The detector is an ultraviolet visible light UV Vis spectrophotometer With a photodiode array of 512 photodiodes and an optical resolution of 1 2 nm the detector operates
46. eter oder Schwefels ure verwenden Durch Methylenchlorid und Dimethylsulfoxid k nnen nichtmetallische Schl uche quellen dadurch wird der Berstdruck des Schlauches erheblich reduziert Warnings that apply to all Waters instruments A 7 A A 8 Attenzione fare attenzione quando si utilizzano tubi in materiale polimerico sotto pressione Indossare sempre occhiali da lavoro protettivi nei pressi di tubi di polimero pressurizzati Spegnere tutte le fiamme vive nell ambiente circostante Non utilizzare tubi eccessivamente logorati o piegati Non utilizzare tubi non metallici con tetraidrofurano THF o acido solforico o nitrico concentrati Tenere presente che il cloruro di metilene e il dimetilsolfossido provocano rigonfiamenti nei tubi non metallici riducendo notevolmente la pressione di rottura dei tubi stessi Advertencia se recomienda precauci n cuando se trabaje con tubos de pol mero sometidos a presi n El usuario deber protegerse siempre los ojos cuando trabaje cerca de tubos de pol mero sometidos a presi n Si hubiera alguna llama las proximidades No se debe trabajar con tubos que se hayan doblado o sometido a altas presiones Es necesario utilizar tubos de metal cuando se trabaje con tetrahidrofurano THF o cidos n trico o sulf rico concentrados Hay que tener en cuenta que el cloruro de metileno y el sulf xido de dimetilo dilatan los tubos no met licos lo que reduce la presi n de ruptura de los t
47. figurable Auto Zero In Calculates an offset value that when added to the sample signal makes the resulting baseline signal zero Changing the rear panel interface connections Via the rear panel display you can open and close certain output connections Opening or closing output connections can be useful when you need to start or stop an injection or troubleshoot system connectivity To change PDA detector rear panel interface connections 1 Inthe console select 2998 PDA Detector from the system tree 2 Inthe PDA detector information window click Troubleshoot gt Rear Panel 3 Inthe PDA Detector Rear Panel dialog box locate a signal connection with an Out label and then click the red or green LED symbol The output signal for the selected signal connection indicates whether it is opened gt or closed Diagnostic tests 4 5 General troubleshooting This section suggests possible causes of errors and recommends troubleshooting actions Keep in mind that the source of apparent detector problems can actually be the chromatography or it can involve other system components Most detector problems are relatively easy to correct If after running the diagnostic functions applicable to your problem and troubleshooting the detector you cannot correct an error condition contact Waters Technical Service department Power surges Power surges line spikes and transient energy sources can adv
48. h accuracy 1 0 nm Wavelength 0 1 nm repeatability Digital filter Variable with data rate Second order filter Fixed 371 nm to 800 nm B 2 Specifications Performance specifications Continued Attribute Noise UV dry Specification 10 AU peak to peak Filter 1 second 30 second segments Wavelength 254 nm Bandwidth 3 6 nm 8 pixel bunch Flow cell analytical 10 mm Data rate 2 Hz Noise UV wet 10 AU peak to peak Filter 1 second 30 second segments Wavelength 254 nm Bandwidth 3 6 nm 38 pixel bunch Flow cell analytical 10 mm Data rate 2 Hz Flow rate 0 5 mL min Solvent water acetonitrile 90 10 Drift UV dry 1000 pAU 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 mm Data rate 2 Hz Linearity lt 5 at 2 0 AU propylparaben series at 257 nm analytical flowcell Data rate 1 2 5 10 20 40 and 80 a Wet testing should be done with water acetonitrile 90 10 to minimize the effects of oxygen at 230 nm Water methanol 90 10 can be substituted with appropriate solvent conditioning B 3 Waters 2998 PDA flow cell specifications Tubing internal diameter Pressure Description Volume uL aa inches rating kt bar psi Inlet Outlet Analytical 9 3 10 0 010 0 01
49. hape differences consider the pairs of spectra shown 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 contrast angle 62 3 Spectra that produce a large spectral contrast angle Normalized absorbance Spectral contrast angle 62 3x Compound A Compound B 22000 24000 260 00 23000 300 00 320 00 340 00 Wavelength nm Spectral contrast angles 5 5 5 6 Spectra with similar shapes In the following figure the absorbance spectra of two compounds A and B are similar They therefore produce a small spectral contrast angle 3 0 Spectra with a small spectral contrast angle Spectral contrast angle 3 0 Compound A Compound B Normalized absorbance 22000 24000 26000 280 00 300 00 320 00 34000 Wavelength nm Differences between spectra of the same compound Small but significant differences between absorbance spectra can result from factors other than the 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 variations in solvent conditions The spectra in the next figure for example show how instrument noise can affect the shape of an absorbance spectrum of one compound at two concentrations one high and
50. he scale A large difference between these two numbers indicates a limited range of miscibility For example some fluorocarbons are immiscible with all the standard solvents and have M numbers of 0 32 Two liquids with dual M numbers are usually miscible with each other A liquid is classified in the M number system by testing for miscibility with a sequence of standard solvents A correction term of 15 units is then either added or subtracted from the cutoff point for miscibility Solvent miscibility C 5 Buffered solvents When using a buffer use a good quality reagent and filter it through a 0 45 um filter Do not leave the buffer stored in the system after use Flush all fluid line pathways with HPLC quality water before shutting the system down and leave distilled water in the system flush with 90 HPLC quality water 10 methanol for shutdowns scheduled to be more than one day Use a minimum of 15 mL for sparge equipped units and a minimum of 45 mL for in line vacuum degasser equipped units Head height Position the solvent reservoirs at a level above the HPLC equipment or on top of the pump or detector with adequate spill protection Minimum tubing bend radius recommendations C 6 When bending tubing consult the following table The bend radius on your tubing should not be less than the arc shown in the table The scale is 1 1 so that the diagram can be used as a template Minimum bend radius for stainl
51. her sampling rate in the instrument method If the value is greater than 50 you should specify a lower sampling rate in the instrument method Example of how baseline noise increases with higher sampling rates PO Par ee Pa sy 5 Hz GREEN yy ma A N 10 Hz UN tion Aalt landen 20 Hz 40 Hz 1 8 2998 PDA Detector Optics Principles Computing absorbance data points The absorbance data points are calculated by the detector and then transmitted to the database MassLynx or Empower The absorbance calculation is Absorbance Logio g r ES S D where S sample energy D dark energy R reference energy elapsed time after start of injection wavelength This value is then filtered as specified Dark current Photodiodes discharge even when they are not exposed to light This discharge is called dark current The dark current is updated by closing the shutter to 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 spectrum is a measure of lamp intensity and mobile phase absorbance at initial conditions The detector records a reference spectrum at the start of each injection The reference spectrum value is calculated using the same filter time constant as the absorbance data Data averaging The data the detec
52. hiol SH 195 1400 Disulfide S S 194 5500 255 400 Bromide Br 208 300 Todide I 260 400 Nitrile C N 160 Acetylide C C 175 180 6000 Sulfone SO2 180 Oxime NOH 190 5000 Azido gt C N 190 5000 Ethylene C C 190 8000 Ketone gt C 0 195 1000 270 285 18 30 Thioketone gt C S 205 strong Esters COOR 205 50 Aldehyde CHO 210 strong 280 300 11 18 Carboxyl COOH 200 210 50 70 Sulfoxide gt S O 210 1500 Wavelength selection C 13 Electronic absorption bands of representative chromophores Continued Chemical max Emax max E max Chromophore gt Configuration nm Lim cm nm L m cm Nitro NO9 210 strong Nitrile ONO 220 230 1000 2000 3800 400 10 Azo N N 285 400 3 25 Nitroso N O 302 100 Nitrate ONO9 270 12 shoulde r Allene C C 9 210 230 21 000 acyclic Allene C C 3 260 35 000 Allene C C 4 300 52 000 Allene C C 5 330 118 000 Allene C C 2 230 260 3000 8000 alicyclic Ethylenic C C C C 219 6 500 Acetylenic Ethylenic C C C N 220 23 000 Amido Ethylenic C C C 0 210 250 10 000 Carbonyl 20 000 Ethylenic C C NO9 229 9 500 Nitro Mobile phase absorbance Willard H H and others Instrumental Methods of Analysis 6th ed Litton Educational Publishing Inc 1981 Reprinted by permission of Wadsworth Publi
53. hose of the type and rating printed on panels adjacent to instrument fuse covers Attention pour viter tout risque d incendie remplacez toujours les fusibles par d autres du type et de la puissance indiqu s sur le panneau proximit du couvercle de la boite fusible de l instrument Vorsicht Zum Schutz gegen Feuer die Sicherungen nur mit Sicherungen ersetzen deren Typ und Nennwert auf den Tafeln neben den Sicherungsabdeckungen des Ger ts gedruckt sind Attenzione per garantire protezione contro gli incendi sostituire i fusibili con altri dello stesso tipo aventi le caratteristiche indicate sui pannelli adiacenti alla copertura fusibili dello strumento Advertencia Para evitar incendios sustituir los fusibles por aquellos del tipo y caracter sticas impresos en los paneles adyacentes a las cubiertas de los fusibles del instrumento EE ATEAK ERARE E A aL ea 97 R EAT EAA E BEIENEE A CECR E EE NEE Eed E E E ER SM ASS SOAR II FARAH Ae WSO Sta Adse EF A Ble F285 MHAIS ON Oe CIT Ea RRT E BO RARE a S BAS BEOEROE 2a KE CEH RAW Warnings that apply to all Waters instruments A 11 Electrical and handling symbols Electrical symbols These can appear in instrument user manuals and on the instrument s front or rear panels Electrical power on Electrical power off Standby C O Direct current Alternat
54. ic acid 0 1 190 50 mM EDTA disodium 1 mM 190 TRIS HCl 20 mM pH 7 0 202 212 pH 8 0 HEPES 10 mM pH 7 6 225 Triton X 100 0 1 240 Hydrochloric acid 0 1 190 Waters PIC Reagent A 200 1 vial liter MES 10 mM pH 6 0 215 Waters PIC Reagent BG 225 1 vial liter Potassium phosphate Waters PIC Reagent B 6 190 monobasic 10 mM 190 low UV 1 vial liter dibasic 10 mM 190 Sodium acetate 10 mM 205 Waters PIC Reagent D 4 190 1 vial liter C 12 Solvent Considerations Wavelength selection for chromophore detection Certain functional groups found in most compounds absorb light selectively These groups known as chromophores and their behavior can be used to categorize the detection of sample molecules The table below lists some common chromophores and their detection wavelengths Amax as well as the molar absorptivity Emax of each group Use this information as a guide to select the optimal operating wavelength for a particular analysis Because of the diversity possible within a given sample scanning over a range of wavelengths may be necessary to determine the best wavelength for a particular analysis Electronic absorption bands of representative chromophores Chromophore pees max mee Amax max Configuration nm Lim cm nm L m cm Ether O 185 1000 Thioether S 194 4600 215 1600 Amine NH2 195 2800 T
55. id the pressure applied to the liquid Changes in the composition temperature or pressure of the mobile phase can all lead to outgassing Effects of intermolecular forces Nonpolar gases N O CO He are more soluble in nonpolar solvents than in polar solvents Generally a gas is most soluble in a solvent with intermolecular attractive forces similar to those in the gas like dissolves like Effects of temperature Temperature affects the solubility of gases If the heat of solution is exothermic the solubility of the gas decreases when you heat the solvent If the heat of solution is endothermic the solubility increases when you heat the solvent For example the solubility of He in H O decreases with an increase in temperature but the solubility of He in benzene increases with an increase in temperature Effects of partial pressure The mass of gas dissolved in a given volume of solvent is proportional to the partial pressure of the gas in the vapor phase of the solvent If you decrease the partial pressure of the gas the amount of that gas in solution also decreases Solvent degassing methods C 8 This section describes the solvent degassing techniques that will help you to attain a stable baseline Degassing your solvent also improves reproducibility and pump performance You can use either of the following methods to degas solvents e Sparging with helium e Vacuum degassing Solvent Considerations Spa
56. ing current Protective conductor terminal Frame or chassis terminal Fuse Recycle symbol Do not dispose in municipal waste EATON A 12 Safety Advisories Handling symbols These handling symbols and their associated text can appear on labels affixed to the outer packaging of Waters instrument and component shipments tt Keep upright Keep dry Fragile CR T K Use no hooks Electrical and handling symbols A 13 A 14 Safety Advisories B Specifications This appendix contains product specifications for the 2998 PDA detector Physical specifications Attribute Specification Height 19 4 cm 7 64 inches Depth 61 cm 24 0 inches Width 34 3 cm 18 5 inches Weight 12 6 kg 27 75 pounds Environmental specifications Attribute Specification Operating temperature 4 to 40 C 89 2 to 104 F Operating humidity lt 95 noncondensing Shipping and storage temperature 30 to 60 C 22 to 140 F Shipping and storage humidity 20 to 85 noncondensing Acoustic noise instrument lt 50 dBA generated Electrical specifications Attribute Specification Protection class Class I Overvoltage category Il Pollution degree 2 B 1 Electrical specifications Continued Attribute Specification Moisture protection Normal IPXO A Line voltages no
57. ioning 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 left of the power LED indicates the lamp status Warning To avoid electric shock do not touch the lamp connector if the lamp LED is flashing red Power off and unplug the detector before touching the lamp connector 2 20 Setting Up the Detector The following table identifies each LED mode with its corresponding detector lamp state Lamp LED indications LED mode and color Description Unlit Indicates the detector lamp is extinguished Constant green Indicates the detector lamp is ignited Flashing green Indicates the detector is initializing or verifying calibration Flashing red Indicates that an error stopped the detector Refer to the console for information regarding the error Constant red Indicates a detector failure that prevents further operation Power off the detector and then power on If the LED is still steady red contact your Waters service representative Shutting down the detector To shut down the detector 1 Ifthe mobile phase contains buffers set the solvent delivery system or pump to deliver 10 mL of HPLC grade water Otherwise set the solvent delivery system or pump to deliver 10 mL of degassed methanol 2 Press the Off On 0 1 switch on the front panel
58. ired break point b Grasp the tubing on both sides of the scribed mark with cloth or plastic covered pliers to prevent marring the surface then gently work the tubing back and forth until it separates c File the tubing ends smooth and straight to minimize dead volume and band broadening Assemble a reusable finger tight fitting at both ends of the column outlet line and at one end of the detector outlet line Reusable finger tight fitting assembly Reusable compression fitting Ferrule End must be straight and smooth to prevent dead volume Tubing Distance determined by the union or column fitting Seat one end 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 Seat the other end of the tubing in the fitting of the detector inlet then tighten the reusable finger tight fitting as in step 5 Seat the end of the detector outlet tubing with the reusable finger tight fitting in the detector outlet fitting finger tighten the fitting until it is snug and then add a quarter turn Insert the other end of the tubing in the waste container Caution To avoid damage to the flow cell avoid pressures approaching its maximum allowable pressure 6895 kPa 69 bar 1000 psi 70 kg cm Plumbing the detector 2 17 8 Slide the 3 8 inch OD Tygon tubing supplied in the startup kit over the barbed d
59. l and lens tissue Do not use abrasive tissue Do not apply excessive pressure 1 Unpack the new lamp from its packing material without touching the bulb 3 8 Maintaining the Detector Inspect the new lamp and lamp housing Position the lamp so that the cut out on the lamp base plate is at the 1 o clock position in line with the alignment pin on the lamp housing and then gently push the lamp forward until it bottoms into position Ensure that it is flush to the optics bench Caution To prevent the lamp from binding and ensure that it is properly seated in the lamp housing alternate between tightening the captive screws and pushing the lamp forward Tighten the two captive screws and then reconnect the lamp power connector Replace the front panel cover Power on the detector and then wait 1 hour for the lamp to warm before resuming operations Tip Cycling power to the detector that is powering off and then powering on the instrument initiates the verification procedures In the console select Maintain gt Change Lamp Change Lamp dialog box Change Lamp Currently Installed Lamp State On and stable Good ignitions 28 Total ignitions 28 Date Installed Serial Number Current Click New Lamp Replacing the lamp 3 9 New Lamp dialog box Serial number of new lamp Cancel 9 Type the serial number for the new lamp see the label attached to the lamp connector wire and then
60. l requires cleaning first try flushing it with solvent To flush the flow cell 1 Select a solvent compatible with the samples and mobile phases that you have been using If you have been using buffers flush with 10 mL of HPLC grade water then flush with 10 mL of a low surface tension solvent such as methanol Tip Ensure that the solvent is miscible with the previous mobile phase 2 Test the lamp energy by performing the Read energy diagnostic test see page 4 3 If the lamp diagnostic test fails and the lamp has not been used more than 2000 hours or 1 year from date of purchase whichever comes first call Waters Technical Service see page 3 2 3 4 Maintaining the Detector Replacing the flow cell Required materials e 1 4 inch flat blade screwdriver e Flow cell To replace the flow cell 1 Power off the detector 2 Stop the solvent 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 cell Lamp Flow cell handle Thumbscrews Flow cell Tip The optional auto purification flow cell has three fittings inlet analytical inlet prep and outlet 5 Remove the flow cell Maintaining the flow cell 3 5 e Use a 1 4 inch flat blade screwdriver to loosen the three thumbscrews on the flow cell assembly s
61. mendations on page C 6 A Warning To avoid chemical hazards always observe Good Laboratory Practices when operating your system Refer to the Material Safety Data Sheets shipped with solvents for handling information Using incompatible solvents can cause severe damage to the instrument and injury to the operator a Caution To prevent contamination wear powder free nonlatex gloves when plumbing the detector Required Materials 5 16 inch open end wrench 0 009 inch 0 23 mm ID stainless steel tubing included in the Startup Kit Stainless steel tubing cutter or scribing file Pliers plastic or cloth covered Compression screw assemblies 3 To make plumbing connections to the detector Oo Caution To avoid damaging the flow cell never cut the 1 32 inch stainless steel flow cell tubing 1 Measure the length of tubing needed to connect the column outlet to the detector inlet Tip Keep the length of this tubing as short as possible to prevent band broadening 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 bubbles from forming in the flow cell 3 Cut the two lengths of tubing as follows 2 16 Setting Up the Detector a Usea Waters 1 16 inch stainless steel tubing cutter or a file with a cutting edge to scribe the circumference of the tubing at the des
62. metimes violated resulting in a deviation from absorbance versus concentration linearity The absorbance of mobile phase can reduce the linear range Absorbance as a function of concentration Absorbance Linear range for analyte Concentration Flow cell operating principles The Waters TaperSlit flow cell used in the 2998 detector renders the detector baseline essentially insensitive to changes in mobile phase refractive index RI RI changes occur during gradient separations or result from temperature or pump induced pressure fluctuations To achieve RI immunity a combination of a spherical mirror a lens at the entrance of the flow cell and a taper to the internal bore of the flow cell 2998 PDA Detector Optics Principles prevents light rays from striking the internal walls of the flow cell The Waters TaperSlit flow cell so called because of the shape of the flow cell exit face matches the shape of the spectrograph slit Compared to a conventional flow cell with a cylindrical shape the 2998 detector achieves higher light throughput for a given spectral resolution with the TaperSlit cell design Comparison of flow cell characteristics Conventional flow cell Resolving spectral data Together with photodiode spacing the detector s 50 um slit determines the intensity and bandwidth of the light that strikes the photodiode array Reducing the bandwidth increases the resolving power of the detecto
63. minal Grounded AC Voltage range 100 to 240 VAC nominal Frequency 50 to 60 Hz Fuse Two fuses 100 to 240 VAC 50 to 60 Hz F 3 15 A 250 V fast blow 5 x 20 mm IEC Power consumption 185 VA nominal a Protection Class I The insulating scheme used in the instrument to protect from elec trical shock Class I identifies a SE level of insulation between live parts wires and exposed conductive parts metal panels in which the exposed conductive parts are con nected to a grounding system In turn 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 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 surface resistivity Degree 2 refers only to normally nonconductive pollution Occasionally however expect a temporary conductivity caused by condensation d Moisture Protection Normal OPO IPXO means that no Ingress Protection against any type of dripping or sprayed water exists The X is a placeholder that identi fies protection against dust if applicable Performance specifications Attribute Specification Wavelength range 190 to 800 nm Optical resolution 1 2 nm Photodiodes 512 Digital resolution 1 2 nm pixel Wavelengt
64. nject Start Inject Start Ground Lamp Off Lamp Off Chart Mark Chart Mark Ground Auto Zero Auto Zero NNN 1 Generating stop flow A Warning Fire and explosion hazard Failure to connect the stop flow output could cause the detector to flood The detector has a programmable switch output that can be controlled by threshold or timed events Making signal connections To generate stop flow make the connections shown in the following table and figure Requirement To automatically stop the chromatographic flow to the system in the event of an error condition or hardware failure the stop flow signal must be connected to the chromatographic pump Detector connections to an Alliance separations module Alliance separations module connector B Pin 4 stop flow red Pin 6 switch 1 red Pin 5 stop flow black Pin 7 switch 1 black 2998 PDA detector connector B Stop flow connections between the Alliance separations module and the detector 2998 PDA detector connector B Waters Alliance connector B 1 Analog 1 2 Analog 1 3 Ground 4 Analog 2 5 Analog 2 Inject Start 1 6 Switch 1 Inject Start 2 7 Switch 1 Ground 3 8 Ground Stop Flow 4 9 Switch 2 Stop Flow 5 10 Switch 2 Hold Inject1 6 Hold Inject1 7 Hold Inject2 8 Hold Inject2 9 Ground 10 Chart Out 11 Chart Out 12 2 12 Setting Up the De
65. nual injector or Inject Start third party manual injector or autosampler Tip If the injector is an e2695 separations module running in Ethernet mode or a 2707 autosampler then the inject start cable should not be connected However if the injector is an e2695 separations module running in IEEE mode then the inject start cable should be connected Connecting to a manual injector If you are using a manual injector with your system connect the signal cables from the rear panel connector on the detector to the injector as shown in the following table Detector connections to a manual injector 2998 PDA detector connector A Manual injector Inject Start red One set of spade lug Inject Start Inject Start black terminals For information on injection trigger signals from a manual injector see page 2 15 Connecting to other instruments 2 8 This section describes signal connections between the detector s rear panel and these items e Waters Alliance Separations Module e Waters 1500 series pump Setting Up the Detector Waters SAT IN Module e Waters or other manual injector e Waters autopurification system Other manufacturers integrator or A D interface device Warning To avoid electrical shock power off instruments being connected before making any electrical connections Requirement To meet the regulatory requirements of immunity from external electrical distur
66. o ensure a gas fail connection is connected to the LC system so that the LC solvent flow stops if the nitrogen supply fails Mass spectrometer shock hazard This warning applies to all Waters mass spectrometers Warning To avoid electric shock do not remove the mass spectrometer s protective panels The components they cover are not user serviceable This warning applies to certain instruments when they are in Operate mode Warning High voltages can be present at certain external surfaces of the mass spectrometer when the 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 voltage warning symbol A 4 Safety Advisories Biohazard warning This warning applies to Waters instruments that can be used to process material that might contain biohazards substances that contain biological agents capable of producing harmful effects in humans Warning Waters instruments and software can be used to analyze or process potentially infectious human sourced products inactivated microorganisms and other biological materials To avoid infection with these agents assume that all biological fluids are infectious observe Good Laboratory Practices and consult your organization s biohazard safety representative regarding their proper use and handling Specific precautions appear in the latest edition of the US National Institutes of Health NIH
67. oise 5 8 optics overview 1 2 plumbing 2 16 rear panel interface connections changing 4 5 displaying 4 4 serial number 2 2 site selection 2 3 starting up 2 19 Index 1 unpacking 2 2 detergents C 18 dimensions 2 4 E EC Authorized Representative vi electrical specifications B 1 electrical symbols A 12 electricity source connecting 2 5 Empower data system connecting 2 14 environmental specifications B 1 equipment guidelines iv A 6 erbium verification performing 4 3 error messages 4 1 eSAT IN module 2 14 Ethernet cable connecting 2 6 external analog data collection device connecting 2 13 F flammable solvents A 4 flow cell clearing bubbles 4 7 comparison 1 5 conditions 2 22 flushing 3 4 installing 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 Index 2 solubility C 7 C 8 generating inject start 2 10 stop flow 2 12 H handling symbols A 13 hardware preparing 3 1 inject start connection 2 7 generating 2 10 signal 2 7 injection trigger signals connecting 2 15 input and output connectors 2 9 installing detector 2 2 lamp 3 8 network guidelines 2 7 intended use v interface connections detector 4 4 ISM classification v L lamp cooling time 3 7 installing 3 8 LED 2 20 4 6 removing 3 7 replacing 3 7 lamp energy reading 4 3 LED lamp 2 20 4 6 monitoring 2 20
68. olvent flush the buffer out of the system with distilled water before you add the organic solvent Solvent miscibility Boilin Miscibilit dere Solvent Mee Point C Number gen a 1 atm M 0 3 N decane 0 92 174 1 29 0 4 Iso octane 0 50 99 2 29 210 0 0 N hexane 0 313 68 7 29 0 0 Cyclohexane 0 98 80 7 28 210 1 7 Butyl ether 0 70 142 2 26 1 8 Triethylamine 0 38 89 5 26 Solvent miscibility C 3 Solvent miscibility Continued Boilin Miscibilit oe Solvent Sei Point C Number 4 mo 1 atm M 2 2 Isopropyl ether 0 33 68 3 220 2 3 Toluene 0 59 100 6 23 285 2 4 P xylene 0 70 138 0 24 290 3 0 Benzene 0 65 80 1 21 280 3 3 Benzyl ether 5 33 288 3 3 4 Methylene chloride 0 44 39 8 20 245 3 7 Ethylene chloride 0 79 83 5 20 3 9 Butyl alcohol 3 00 117 7 3 9 Butanol 3 01 177 7 15 4 2 Tetrahydrofuran 0 55 66 0 17 220 4 3 Ethyl acetate 0 47 RO 19 260 4 3 1 propanol 2 30 97 2 15 210 4 3 2 propanol 2 35 117 7 15 4 4 Methyl acetate 0 45 56 3 15 17 260 4 5 Methyl ethyl ketone 0 43 80 0 17 330 4 5 Cyclohexanone 2 24 155 7 28 210 4 5 Nitrobenzene 2 03 210 8 14 20 4 6 Benzonitrile 1 22 191 1 15 19 4 8 Dioxane 1 54 101 3 17 220 5 2 Ethanol 1 20 78 3 14 210 5 3 Pyridine 0 94 115 3 16 305 5 3 Nitroethane 0 68 114 0
69. ontrast algorithm uses absorbances from multiple wavelengths to form a vector in an n dimensional vector space where n is the 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 mathematically to compute the spectral contrast angle As with the two wavelength comparison a spectral contrast angle of zero in n dimensional space means that all ratios of absorbances at corresponding wavelengths match Conversely if any comparison of ratios does not match the corresponding vectors point in different directions Spectral contrast angles 5 4 Spectra of identical shape have vectors that point in the same direction Spectra of varying shapes have vectors that point in different directions The angle between the two vectors of any two spectra the spectral contrast angle expresses the difference in direction between the spectral vectors of two spectra A spectral contrast angle can vary from 0 to 90 A spectral contrast angle approaching 0 indicates little shape difference between the compared spectra Matching a spectrum to itself produces a spectral contrast angle of exactly 0 The maximum spectral contrast angle 90 indicates that the two spectra do not overlap at any wavelength Spectral Contrast Theory To illustrate the relationship between the spectral contrast angle and spectral s
70. or s guide accompanies the instrument consult the instrument s online Help system for calibration instructions Quality control Routinely run three QC samples that represent subnormal normal and above normal levels of a compound Ensure that QC sample results fall within an acceptable range and evaluate precision from day to day and run to run Data collected when QC samples are out of range might not be valid Do not report these data until you are certain that the instrument performs satisfactorily ISM classification ISM Classification ISM Group 1 Class B This classification has been assigned in accordance with CISPR 11 Industrial Scientific and Medical ISM instruments requirements Group 1 products apply to intentionally generated and or used conductively coupled radio frequency energy that is necessary for the internal functioning of the equipment Class B products are suitable for use in both commercial and residential locations and can be directly connected to a low voltage power supply network EC authorized representative Waters Corporation Floats Road Wythenshawe Manchester M23 9LZ United Kingdom 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 vi Table of Contents CO Py Pil Te TE ii KEE ii Customer COMMENTS esserne ranen ano SEa N E ATAD ETEA NEEESE EEEa iii
71. or 2 18 Using a cuvette 2 21 2 1 Before you begin Requirement To install the 2998 PDA detector you should know how in general to set up and operate laboratory instruments and computer controlled devices and also how to handle solvents Before installing the detector ensure that e the required components are present none of the shipping containers or unpacked items are damaged Unpacking and inspecting 2 2 The detector shipping carton contains Certificate of Structural Integrity 2998 PDA detector Waters 2998 PDA Detector Operator s Guide this document Startup kit Release notes To unpack the detector and flow cell 1 Check the contents of the shipping cartons 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 of the cartons immediately contact the shipping agent 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 or Waters corporate headquarters in Milford Massachusetts USA or visit http www waters com and click Offices For complete information on reporting shipping damages and submitting claims see Waters Licenses Warranties and Support Services Make sure the instrument serial
72. osed to the vacuum the more dissolved gases are removed Two factors affect the amount of time the solvent is exposed to the vacuum Flow rate At low flow rates most of the dissolved gas is removed as the solvent passes through the vacuum chamber At higher flow rates lesser amounts of gas per unit volume of solvent are removed Surface area of the degassing membrane The length of the degassing membrane is fixed in each vacuum chamber To increase the length of membrane you can connect two or more vacuum chambers in series The in line degasser is available as an option or factory installed in the Waters 2695 Separations Module XE model Wavelength selection This section includes UV cutoff ranges for common solvents common mixed mobile phases chromophores UV cutoffs for common solvents The table below shows the UV cutoff the wavelength at which the absorbance of the solvent is equal to 1 AU for some common chromatographic solvents Operating at a wavelength near or below the cutoff increases baseline noise because of the absorbance of the solvent UV cutoff wavelengths for common chromatographic solvents Solvent Ra Solvent roe tua 1 Nitropropane 380 Ethylene glycol 210 2 Butoxyethanol 220 Iso octane 215 Acetone 330 Isopropanol 205 Acetonitrile 190 Isopropyl chloride 225 C 10 Solvent Considerations UV cutoff wavelengths for common chromatographic solvents
73. pectra of two compounds Ab 245 Compound A 2 2 P Ab257 AD 245 0 40 07 245 nm Compound B AD oer 257 nm Compound A Compound B D gt lt 0 20 0 00 220 00 240 00 260 00 280 00 300 00 320 00 340 00 nm Representing spectra as vectors The spectral contrast algorithm uses vectors to quantify differences in the shapes of spectra converting baseline corrected spectra to vectors and then comparing the vectors Spectral vectors have two properties 5 2 Spectral Contrast Theory e Length Proportional to analyte concentration Direction Determined by the relative absorbance of the analyte at all wavelengths its absorbance spectrum Direction is independent of concentration for peaks that are less than 1 0 absorbance units AU across the collected wavelength range Vector direction contributes to the identification of a compound because the direction is a function of the absorbance compound s spectrum The ability of spectral vectors to differentiate compounds depends on the resolution of spectral features As both wavelength range and spectral resolution increase the precision of a spectral vector 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 include wavelengths where there is no analyte absorbance Vectors derived from two wavelengths The sp
74. r As a result similar spectra are more effectively distinguished The grating images the slit onto the photodiode array The angle of diffraction from the grating determines the wavelength that strikes a particular photodiode in the array The following figure shows an absorbance spectrum of benzene Note that the wavelength resolution is sufficient to resolve five principal absorption peaks Resolving spectral data 1 5 Benzene spectrum at different resolutions Absorbance 230 00 250 00 270 00 nm Measuring light at the photodiode array The detector measures the amount of light striking the photodiode 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 Light striking a photodiode discharges the diode The magnitude of the discharge depends on the amount of light striking the photodiode 1 6 2998 PDA Detector Optics Principles Photodiodes discharged by light Sample in flow cell absorbs at specific Q Deuterium lamp wavelengths Grating cm Flow cell Light from grating dispersed onto diodes Err SE Mirror The detector measures the amount of charge required to recharge each photodiode The charge is proportional to the amount of light transmitted through the flow cell over the interval specified
75. r calibration 1 Select a solvent compatible with the samples and mobile phases that you have been using If you have been using buffers flush with 10 mL of HPLC grade water then flush with 10 mL of a low surface tension solvent such as methanol Tip Ensure that the solvent is miscible with the previous mobile phase 2 Inthe console select 2998 PDA Detector from the system tree 3 Inthe PDA detector information window click Maintain gt Verify calibration gt Start The test time appears in the Run Time bar graph 4 When the test ends the Results pane appears Verify that the detector passed the test Diagnostic Tests and Troubleshooting Rule The maximum deviation must be within 1 nm of the standard calibration to pass Tip If the test fails flush the flow cell and run the verify detector calibration procedure again Click Close Reading lamp energy Recommendation Impurities in the flow cell may affect reading lamp energy Ensure that the flow cell is clean before you read lamp energy To read the lamp energy 1 2 In the console select 2998 PDA Detector from the system tree In the PDA detector information window click Maintain gt Read energy gt Read The Read Energy dialog box appears Click Close Performing the erbium calibration Recommendation Impurities in the flow cell may affect wavelength calibration Ensure that the flow cell is clean before you perform calibration Before
76. r spare parts details see the Waters Quality Parts Locator on the Waters web site s Services Support page or the 2998 PDA Detector spare parts list part number 71500121906 Routine Maintenance The 2998 PDA detector requires minimal routine maintenance To achieve optimal performance follow these steps 1 Replace solvent reservoir filters in your HPLC system regularly 2 Filter and degas solvents to prolong column life reduce pressure fluctuations and decrease 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 of the solvent lines and flow cell e Damage to the components e Microbial growth Routine Maintenance 3 3 Maintaining the flow cell The flow cell requires maintenance when the reference spectrum changes the cell fluid leaks out of the drain tube the detector cannot initialize but the lamp is in good condition e the detector causes high backpressure Tip Conditions other than a dirty flow cell can cause decreased lamp intensity For more information refer to Chapter 4 Flow cell maintenance consists of e flushing the flow cell removing the flow cell disassembling and cleaning the flow cell installing the flow cell assembly Flushing the flow cell Required Materials HPLC grade water HPLC grade methanol If the flow cel
77. rain fitting located on the drip tray and route it to a suitable waste container Making the gas supply connection The detector can be connected to a nitrogen source to improve operation at lower wavelengths Requirement Only use a constant supply 28 to 41 kPa 28 to 41 bar 4 to 6 psi of dry oil free filtered nitrogen Warning Do not use gases that would allow the combustion of combustible solvents Always use inert gases To make the gas supply connection 1 Connect one end of the plastic tubing to the nitrogen source Tip The nitrogen source may require an adapter in order to fit the plastic tubing 2 Connect the other end of the nitrogen fitting on the rear panel of the detector Starting up and shutting down the detector A Warning Always observe Good Laboratory Practices when you use this equipment and when you work with solvents and test solutions Know the chemical and physical properties of the solvents and test solutions you use See the Material Safety Data Sheet for each solvent and test solution in use Using incompatible solvents can cause severe damage to the instrument and injury to the operator The detector startup procedure takes less than one minute Once completed you should allow the detector to warm up for at least one hour before running an analysis Follow the procedures in this section to ensure reliable detector performance 2 18 Setting Up the Detector Starting up the dete
78. rements Before beginning a measurement using the cuvette 1 Fill the flow cell by flushing with 10 mL of the same mobile phase that you are going to use with the cuvette measurement 2 To ensure a clear and clean cuvette wipe the clear portion of the cuvette with low lint nonabrasive tissue wipes 2 22 Setting Up the Detector Cuvette measuring procedure To begin a cuvette measurement 1 Remove the detector front left panel cover 2 To remove the cuvette holder slide it toward you Cuvette holder 3 With the spring guide facing you gently insert the cuvette containing eluent up and under the guide with the cap facing upward into the holder and a frosted side of the cuvette facing up Refer to the figure on page 2 22 Recommendations e Ensure that you have enough liquid 8 mL in the cuvette so that when it is inserted into the holder you can see liquid through the cuvette holder aperture that is the liquid completely covers the aperture e Because the cuvette holder is angled use your thumb or forefinger to ensure the cuvette is secure in the slot and does not slide forward e Ensure that it does not become dislodged when you are replacing the cuvette holder 4 Gently guide the cuvette holder back into the flow cell assembly until it sits in position securely Using a cuvette 2 23 5 Reinstall the detector front left
79. res and the guidelines presented in this section Applicable symbols Symbol Waters Corporation 34 Maple Street Milford MA 01757 U S A Definition Manufacturer EC REP Waters Corporation Floats Road Wythenshawe Manchester M23 9LZ United Kingdom Authorized representative of the European Community CE Confirms that a manufactured product complies with all applicable European Community directives ABN 49 065 444 751 Australia C Tick EMC Compliant Confirms that a manufactured product complies with all applicable United States and Canadian safety requirements Consult instructions for use Audience and purpose This guide is intended for personnel who install operate and maintain the Waters 2998 Photodiode Array PDA detector Intended use of the 2998 Photodiode Array detector Waters designed the 2998 Photodiode Array detector to analyze and monitor various types of compounds The 2998 PDA detector is for research use only Calibrating To calibrate LC systems follow acceptable calibration methods using at least five standards to generate a standard curve The concentration range for standards should include the entire range of QC samples typical specimens and atypical specimens When calibrating mass spectrometers consult the calibration section of the operator s guide for the instrument you are calibrating In cases where an overview and maintenance guide not operat
80. rging Sparging removes gases from solution by displacing dissolved gases in the solvent with a less soluble gas usually helium Well sparged solvent improves pump performance Helium sparging brings the solvent to a state of equilibrium which may be maintained by slow sparging or by keeping a blanket of helium over the solvent Blanketing inhibits reabsorption of atmospheric gases Sparging can change the composition of mixed solvents Vacuum degassing The in line vacuum degasser operates on the principle of Henry s Law to remove dissolved gases from the solvent Henry s Law states that the mole fraction of a gas dissolved in liquid is proportional to the partial pressure of that gas in the vapor phase above the liquid If the partial pressure of a gas on the surface of the liquid is reduced for example by evacuation then a proportional amount of that gas comes out of solution Vacuum degassing can change the composition of mixed solvents Solvent degassing considerations Select the most efficient degassing operation for your application To remove dissolved gas quickly consider the following Sparging Helium sparging gives stable baselines and better sensitivity than sonication in a detector and prevents reabsorption of atmospheric gases Use this method to retard oxidation when you are using tetrahydrofuran or other peroxide forming solvents Solvent degassing methods C 9 Vacuum Degassing The longer the solvent is exp
81. sevsecseseecaveeseasecesaes 3 4 Piss ihe llow E E 3 4 viii Table of Contents E the low WE 3 5 Replacing Che A arrana aaa a aaa aaaea aaaea aa aa aa Saaana danced EEE E ESEESE 3 7 Replacing the USES aeaaea aaa aaaea aaaea aaa aaaea ee deg 3 10 4 Diagnostic Tests and Troubleshooting sssssssssssssesseesececeeeecossesssssse 4 1 KEN oe d E 4 2 Vveriyine deteclor airs e EE 4 2 Roading EE 4 3 Performing the erbium calibration cccccccceesssccsseccecceeeeseeceeeeeeeeeeeaeeeseeeas 4 3 Reading the calibration constants E 4 4 Displaying PDA detector rear panel interface connections sssessesseese 4 4 Changing the rear panel interface Conmnectons 4 5 General troubleshooting ssssscssscssssssccsscsssccscccccccsccecccecccsccecccecceeceecceeees 4 6 Poner BTSs icra Ie 4 6 Clearing bubbles from the flow cell oo cccccccceccceccecccceccecceecececcssceseeens 4 7 Detector troubleshooting esssessseseesseessreseeerrerrerrererecererreeereereereerereeeeereeseeess 4 7 5 Spectral Contrast Theory sisisissiscasccssisccacsssancssceveissasnsaacacccasscscacesasascesces 5 1 Comparing absorbance spectra ccccccccccccccccceccsccceccceccscccscccscessccsscescccesees 5 2 Representing spectra as vectors ssesessseossssesessecocossesessececoocososseseeeocossssesee 5 2 Vectors derived from two wavelengths sssssssssssssessserrssssssoserrssssssssrrsreeess 5 3 Vectors derived from multiple wavelengthe 5
82. shing Co Belmont California 94002 This section lists the absorbances at several wavelengths for frequently used mobile phases Choose the mobile phase carefully to reduce baseline noise The best mobile phase for your application is one that is transparent at the chosen detection wavelengths With such a mobile phase ensure that any C 14 Solvent Considerations absorbance is due only to the sample Absorbance by the mobile phase also reduces the linear dynamic range of the detector by the amount of absorbance the autozero function cancels or autozeroes out Wavelength pH and concentration of the mobile phase affects its absorbance Examples of several mobile phases are given in the table below Tip The absorbances in 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 Ion 215 220 230 240 250 260 280 Solvents Acetonitrile 0 05 0 03 0 02 0 01 0 01 lt 0 01 Methanol 2 06 1 00 0 53 0 37 0 24 0 11 0 05 0 02 lt 0 01 not degassed Methanol 1 91 10 76 0 385 0 21 10 15 10 06 0 02 lt 0 01 degassed Isopropanol 1 80 0 68 0 34 0 24 0 19 0 08 004 0 03 0 02 0 02 Unstablized 2 44 2 57 2 81 1 80 1 54 0 94 0 42 0 21 0 09 008 tetrahydro furan THF fresh Unstablized gt 2 5 gt 2 5 gt 2 5 gt 2 5 gt 2 5 gt 2 5 gt 2
83. t you expect from our documentation so that we can continuously improve its accuracy and usability We seriously consider every customer comment we receive You can reach us at tech_comm waters com Contacting Waters Contact Waters with enhancement requests or technical questions regarding the use transportation removal or disposal of any Waters product You can reach us via the Internet telephone or conventional mail Waters contact information Contacting medium Internet Information The Waters Web site includes contact information for Waters locations worldwide Visit www waters com Telephone and fax From the USA or Canada phone 800 252 HPLC or fax 508 872 1990 For other locations worldwide phone and fax numbers appear in the Waters Web site Conventional mail Waters Corporation 34 Maple Street Milford MA 01757 USA Safety considerations Some reagents and samples used with Waters instruments and devices can pose chemical biological and radiological hazards You must know the potentially hazardous effects of all substances you work with Always follow Good Laboratory Practice and consult your organization s safety representative for guidance Safety advisories Consult Appendix A for a comprehensive list of warning and caution advisories Operating this instrument When operating this instrument follow standard quality control QC procedu
84. tector Connecting to a chart recorder or external analog data collection device To send an analog output signal from the detector to a chart recorder make the connections shown in the following table and figure 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 black Tip For this connection do not use the cable shield Analog output connections to a chart recorder 2998 PDA detector connector B Analog 1 Analog 1 Ground Analog 2 Analog 2 Switch 1 Switch 1 Ground Switch 2 Switch 2 eleleie La v2 Chart recorder connectors A OO JO Om ob ll aol oe CH l Making signal connections 2 13 2 14 Connecting to an Empower or MassLynx data system using an eSAT IN module To send an integrator analog output signal 0 1 to 2 1 V from the detector to an Empower or MassLynx system through a two channel SAT IN Module make the connections shown in the following table and figure Detector connections to the eSAT IN module SAT IN module connector CHANNEL 1 2998 PDA detector connector B Pin 1 signal out white Pin 2 signal out black Analog output connections to the eSAT IN module eSAT IN module Waters e SAT IN HANNEL 2 Injector 2998 PDA detector
85. thority to operate the equipment Important Toute modification sur cette unit n ayant pas t express ment approuv e par l autorit responsable de la conformit la r glementation peut annuler le droit de l utilisateur exploiter l quipement Achtung Jedwede Anderungen oder Modifikationen an dem Ger t ohne die ausdr ckliche Genehmigung der f r die ordnungsgem e Funktionstiichtigkeit verantwortlichen Personen kann zum Entzug der Bedienungsbefugnis des Systems f hren Avvertenza qualsiasi modifica o alterazione apportata a questa unit e non espressamente autorizzata dai responsabili per la conformit fa decadere il diritto all utilizzo dell apparecchiatura da parte dell utente Atencion cualquier cambio o modificaci n efectuado en esta unidad que no haya sido expresamente aprobado por la parte responsable del cumplimiento puede anular la autorizaci n del usuario para utilizar el equipo ES REA BEN I EE AARE A EAT SB IB ul GE E EEH A TE RRE RAS fii RERI o VER A n LUE RIT I HAVAITA ETT I RB ON FY ESS BH Te RR WEARS IEEE SO nS ESS HONE SAAS Opel Sol ROl Ol SAS WA SE HAS SE 0 SIE SSE zc AS AD Gs ASS US L C TERR RABID 5 DAME ZR AGB amp Se IT PICA OBR OBE 1T 9 L ARO a F UC ORR PEC 72S REEDS OES Safety Advisories A Warning Use caution when working with any polymer
86. tializing No corrective action required Wait until initialization completes both lights constant green Diagnostic Tests and Troubleshooting PDA detector troubleshooting Continued Symptom Lamp light flashes red and power light constant green Possible cause Failed startup diagnostic tests Corrective action Reseat and check alignment of flow cell see page 3 5 Flush the flow cell see page 3 4 Dirty flow cell causing shutter diagnostic test to fail Flush the flow cell see page 3 4 Insufficient energy reaching photodiode array 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 60 cm length of 0 009 inch 0 23 mm ID tubing connected to the detector waste outlet Weak lamp Replace the lamp see page 3 7 Shutter failure message Shutter failed 1 Clear bubbles from the flow cell see page 4 7 2 Power the PDA detector off and on again General troubleshooting 4 9 PDA detector troubleshooting Continued Symptom Solvent in drain line Possible cause Leak from flow cell gasket Corrective action Replace the flow cell see page 3 5 Leak from flow cell inlet and outlet fittings Check fittings for overtightening or undertightening and
87. tion of effects can produce slight departures about 1 from Beer s law because of photometric error Although photometric errors at this level can negligibly affect quantitation they can nevertheless be a significant source of spectral inhomogeneity To minimize the effects of photometric error for all spectral contrast operations the maximum spectral absorbance of a compound should 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 phase absorbance see Appendix C See also For more information about the effects of the photometric error curve refer to Principles of Instrumental Analysis 3rd ed by Douglas A Skoog Saunders College Publishing 1985 pp 168 172 Solvent changes As long as solvent concentration and composition do not change isocratic operation background absorbance if any by the solvent remains constant However change in solvent pH or composition such as that which occurs in gradient operation can affect the intrinsic spectral shape of a compound See the figure Effects of pH on the absorbance spectrum of p aminobenzoic acid on page 5 9 Threshold angle 5 8 In addition to computing spectral contrast angles the spectral contrast algorithm also computes a threshold angle The threshold angle is the maximum spectral contrast angle between spectra that can
88. tor reports to the database Empower or MassLynx can be the average of a number of data points After calculating absorbance the detector averages absorbance values based on the requested spectral resolution Computing absorbance data points 1 9 Averaging spectral resolution The detector can simultaneously collect two types of data channels spectra 8D and chromatograms 2D For best results in library matching and peak 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 for an analyte corresponds to a peak s lambda max increasing bandwidth tends to decrease peak height while reducing baseline noise and linear dynamic range Tip A resolution of 3 6 nm is effective for many analytes 1 10 2998 PDA Detector Optics Principles Resolution comparison for anthracene Absorbance 3 20 3 30 3 40 3 50 3 60 3 70 3 80 3 90 4 00 4 10 4 20 252 nm at 1 2 nm height 0 53 252 nm at 6 0 nm height 0 45 SE 252 nm at 12 0 nm height 0 35 Averaging chromatographic sampling rate Sample rate is the number of data points acquired per second The number of times a given pixel is read during the sample rate 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 1000 msec second 20 samples second
89. ubos Ba HEA RIEV ERRA HERR MNE EA PASH HROBE JI AREA WE CD RERE o TE BASEL TA HI KK o AN EAE E REIER eg o E E KEE E HEN N BET AAR sat Foe HE aR mg ENC ee A PS He ik o MIE E SN IE Se E EP AU Ne KARE REY TER EY i 3 Safety Advisories Ze ARTI EHER Gren SRA Hs HAYES re Be GC EG IR TER BAGEL Dr I KIA o SB CO A It Pe EAS HH YP AS BERE AE Sie JRE Be PSEA a EI TAS DEE ITA r BET fee A A APER FE il SAFARGA E KOK BARE RRINE REZ o AD Jg Sell 82 FAS SLHE FANA AG Ball FS SAAN SA Ss CaS sch i 249 87 8 2F DAA ASHA Su DO SSE ASA OMA Hlas Nonmetallic FSS HE26 amp et Tetrahydrofuran THF 5 ss ae Fe Sha SA ASotA NAS amp st OS al Methylene eee g C O Es A E Dimethyl sulfoxide Was S35 Eq SO re yas AA SAAB s ALn RANA a SY Fa TER La EELT KAY LIESE et ONE EE d leide UPUREA EL CPU VES ICHSKEWULTCK Ei BUX BBLR EMH Bok F aT re LZRVY CK FAV Fe BF a TIC FRAE FeZ ATHA SRR EOR E Ke LI kierze ZG ice KEV ISIEAT Lon d TA Fit HEEF a FORE a El TRAD bY ZORA Fa TRD TEEI TREL ET Warnings that apply to all Waters instruments A 9 Warning The user
90. vent buffered solvents C 6 general considerations C 2 C 3 guidelines C 2 Index 3 miscibility C 38 C 5 reservoirs C 6 UV cutoff C 10 C 12 viscosity considerations C 7 solvent angle photometric error effects 5 8 solvent changes 5 8 spare parts 3 3 sparging C 9 specifications electrical B 1 environmental B 1 flow cell B 4 operational B 2 physical B 1 spectra absorbance comparing 5 2 derived vectors 5 4 differences between 5 6 different shapes 5 5 representing as vectors 5 2 similar shapes 5 6 spectral shape differences 5 7 vectors 5 2 spectral contrast angle 5 4 derived vectors 5 4 spectral shape differences 5 7 vectors 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 Index 4 handling A 13 warning A 2 T TaperSlit flow cell 1 4 threshold angle 5 8 transient energy 4 6 troubleshooting contacting Waters 4 6 detector 4 7 diagnostic functions 4 1 tubing bend radius minimum C 6 cutting 2 16 U undesirable effects shape differences 5 7 V vacuum degassing See degassing vectors derived from multiple wavelengths 5 4 derived from two wavelengths 5 3 spectra representing 5 2 spectral contrast 5 2 verifying calibration 4 2 W warning symbols A 2 A 6 wavelength derived vectors 5 4 mobile phase absorbances C 14 reference 1 13 selection C 10 C 12
91. with a 0 45 um filter Degas and or sparge the solvent Stir the solvent Keep in a place free from drafts and shock Water Use water only from a high quality water purification system If the water system does not deliver filtered water filter it through a 0 45 um membrane filter before use Using buffers When you use buffers dissolve salts first adjust the pH then filter to remove insoluble material C 2 Solvent Considerations Tetrahydrofuran When using unstabilized tetrahydrofuran ensure that your solvent is fresh Previously opened bottles of tetrahydrofuran contain peroxide contaminants which cause baseline drift Warning Tetrahydrofuran contaminants peroxides are potentially explosive if concentrated or taken to dryness Solvent miscibility Before you change solvents refer to the table below to determine the miscibility of the solvents to be used When you change solvents be aware that changes involving two miscible solvents may be made directly Changes involving two solvents that are not totally miscible for example from chloroform to water require an intermediate solvent such as isopropanol temperature affects solvent miscibility If you are running a high temperature application consider the effect of the higher temperature on solvent solubility buffers dissolved in water can precipitate when mixed with organic solvents When you switch from a strong buffer to an organic s
92. you begin this procedure your detector must be set up and configured as described in Chapter 2 During the start up verification sequence the PDA detector performs an erbium calibration You can also start this procedure manually To start a manual erbium verification 1 2 In the console select 2998 PDA Detector from the system tree In the PDA detector information window click Troubleshoot gt Erbium calibration The Erbium Calibration dialog box appears Click Optimize Exposures Click Start Result The detector moves the filter into the erbium position locates the 256 5 378 9 and 521 4 nm erbium absorbance peaks and locates the 656 1 and 486 1 nm deuterium emission lines Diagnostic tests 4 3 Rule The maximum deviation must be within 1 nm to pass 5 Click Stop The erbium filter moves back to original position O Caution This procedure can affect spectral library matching and peak purity calibrations You might need to reacquire spectral libraries each time you perform any calibration procedure See Chapter 5 for more information 6 Click Save gt OK gt Close Reading the calibration constants To read the calibration constants 1 Inthe console select 2998 PDA Detector from the system tree 2 Click Troubleshoot gt Calibration constants The Calibration Constants dialog box appears 3 Click Read 4 Click Close Displaying PDA detector rear panel interface connections You

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