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Waters 2996 PDA Detector

1. 2 Lift up the front panel cover and pull it away from the chassis 3 Open lamp secure panel Alignment Notch Lamp Power Mounting Connector Screws Lamp Usage Indicator TP01466 Figure 3 4 Lamp Power Connector and Mounting Screws 26 Maintenance 4 Usea slotted head screwdriver to unscrew the two mounting screws 5 Grip the metal base of the lamp pull the lamp out and set it aside Do not pull the wires to remove the lamp Carefully unpack the replacement lamp 7 While wearing powder free gloves and holding the lamp by its base position the lamp so that the notch in the base aligns with the positioning pin in the optics bench 8 Insert the lamp and secure it with the two screws Make sure that the lamp base is flush against the lamp housing 9 Reconnect the lamp power connector Figure 3 4 10 Secure the lamp access panel 11 Install the front panel cover 12 Reconnect the power cord and power on the 2996 Detector 3 3 Replacing the Fuses Replace the fuses under the conditions indicated in the troubleshooting table Section 2 1 Diagnostics The 2996 Detector requires two 2 A 250 V fuses 5 mm x 20 mm Caution To avoid electrical hazards power off the 2996 Detector and disconnect the power cord before you perform the following procedure To replace the two fuses in the 2996 Detector 1 Poweroff the 2996 Detector and remove the power cord 2 Locate
2. The Spectral Contrast algorithm uses vectors to characterize spectra Figure 5 2 To understand the vector principle consider two vectors Figure 5 2 based on the spectra depicted in Figure 5 1 0 4 Compound B 0 3 0 2 Compound A AU at 257 nm 0 0 1 0 2 0 3 0 4 AU at 245 nm Figure 5 2 Plotting Vectors for Two Spectra The axes in Figure 5 2 are in absorbance units at the two wavelengths used to calculate the absorbance ratio shown in Figure 5 1 The head of the vector for Compound A is at the intersection of the absorbance values for Compound A at the two wavelengths represented by each axis The other vector is similarly derived for the spectrum of Compound B The vector for Compound B points in a direction different from that of the vector for Compound A The difference in direction which reflects the difference in the absorbance ratios of the two compounds at wavelengths 245 nm and 257 nm is called the Spectral Contrast angle A Spectral Contrast angle e g 0 in Figure 5 2 greater than zero indicates a shape difference between spectra Section 5 3 Spectral Contrast Angles The length of the vector is proportional to the concentration 5 2 2 Vectors Derived from Multiple Wavelengths When absorbance ratios are limited to two wavelengths the chance that two different spectra will have the same absorbance ratio is much greater than if comparison is made using absorbance ratios at many wavelengths Therefore the
3. Height 8 5 in 22 cm Weight 31 5 Ibs 14 3 kg Wavelength range 190 to 800 nm Wavelength accuracy 1 nm Linearity range 5 at 2 0 AU propylparaben at 256 nm Spectral resolution 1 2 nm Baseline noise 1 5 x 10 AU peak to peak dry at 254 nm Drift 1 x 10 AU hour at 254 nm after warmup AT lt 1 C per hour Flow cells Pathlength mm Tubing ID Standard 10 0 009 in Semi preparative 3 0 040 in Variable path flow cell 0 15 to 3 0 004 in Microbore 3 0 005 in Inert 10 0 010 in Autopurification 0 5 0 009 in A Inlet 0 020 in P Inlet 0 040 in Common Outlet Per ASTM 685 79 Detector Specifications 51 52 Detector Specifications Appendix B Spare Parts The spare parts listed in Table B 1 are recommended for customer installation Damage incurred by performing unauthorized work on your 2996 Detector may invalidate certain watranties Table B 1 Spare Parts Item Part Number Flow cell standard WAT057919 Flow cell semi preparative WAT057463 Flow cell microbore WAT057462 Flow cell inert WAT057461 Flow cell Autopurification 289000612 Variable pathlength flow cell WAT057664 Gasket flow cell 2 WAT057924 Belleville washer 2 WAT057925 Lens mount and lens 2 WAT057923 Semi prep lens kit WAT057968 Deuterium lamp WAT052586 PM Kit Fuse fast 4A 250 V 5 x 20 mm WAT057337 Waters Erbium Perchlorate Wavelength WAT042885 Accuracy Solution Waters Absorbance Detector
4. Linearity WAT042881 Solution Spare Parts 53 54 Spare Parts Appendix C Mobile Phase Absorbance This appendix lists the absorbances at several wavelengths for commonly used mobile phases Choose your mobile phase carefully to reduce baseline noise The best mobile phase for your application is one that is transparent at the chosen detection wavelengths Such a mobile phase ensures that any 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 that is autozeroed out Wavelength pH and concentration of the mobile phase will affect its absorbance Examples of several mobile phases are provided in Table C 1 Table C 1 Mobile Phase Absorbance Measured Against Air or Water Absorbance at Specified Wavelength nm 200 205 210 215 220 230 240 250 260 280 Solvents Acetonitrile 0 05 0 03 0 02 0 01 0 01 lt 0 01 Methanol not 2 06 1 00 0 53 0 37 0 24 0 11 0 05 0 02 lt 0 01 degassed Methanol 1 91 0 76 0 35 0 21 0 15 0 06 0 02 lt 0 01 degassed Isopropanol 1 80 0 68 0 34 0 24 0 19 0 08 0 04 0 03 0 02 0 02 Unstablized 2 44 2 57 2 31 1 80 1 54 0 94 0 42 0 21 0 09 0 05 Tetrahydrofuran THF fresh Unstablized gt 2 5 gt 2 5 gt 2 5 gt 2 5 gt 2 5 gt 2 5 gt 2 5 gt 2 5 2 5 1 45 Tetrahydrofuran THF old Mobile Phase Absorbance 55
5. Table C 1 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 1 2 61 2 63 2 61 2 43 2 17 0 87 0 14 0 01 lt 0 01 Hydrochloric 0 11 0 02 lt 0 01 acid 0 1 Phosphoric acid lt 0 01 0 1 Trifluoroacetic 1 20 0 78 0 54 0 34 0 22 0 06 0 02 lt 0 01 acid Diammonium 1 85 0 67 0 15 0 02 0 01 phosphate 50 mM Triethylamine 2 33 2 42 2 50 2 45 2 37 1 96 0 50 0 12 0 04 0 01 196 Buffers and Salts Ammonium 1 88 0 94 0 53 0 29 0 15 0 02 0 01 acetate 10 mM Ammonium 0 41 0 10 0 01 0 01 bicarbonate 10 mM EDTA disodium 0 11 0 07 0 06 0 04 0 03 0 03 0 02 0 02 0 02 0 02 1 mM HEPES 10 mM 2 45 2 50 2 37 2 08 1 50 10 29 0 03 lt 0 01 pH 7 6 MES 10 mM 2 42 2 38 1 89 0 90 045 0 06 lt 0 01 pH 6 0 56 Mobile Phase Absorbance Table C 1 Mobile Phase Absorbance Measured Against Air or Water Continued Absorbance at Specified Wavelength nm 200 205 210 215 220 230 240 250 260 280 Potassium 0 03 lt 0 01 phosphate monobasic KH2PO 10 mM Potassium 0 53 0 16 0 05 0 01 0 01 phosphate dibasic KoHPO 10 mM Sodium acetate 1 85 0 96 0 52 0 30 0 15 0 03 0 01 10 mM Sodium chloride 2 00 1
6. Vectors Derived from Two Wavelengths i 43 5 2 2 Vectors Derived from Multiple Wavelengths 43 5 3 Spectral Contrast Angles ica aa alora 44 5 4 Undesirable EffeCtS cu rionali oie serpit pica ina 47 DATE Detector NOISE tacita paola belle een 47 54 2 Photometric EE 48 5 443 Solvent Changes nia 48 O A lee 48 xii Table of Contents Appendix A Detector Specifications sse 51 Appendix B Spare Parts ee SE 53 Appendix C Mobile Phase Absorbance nooo nono nono erne rent rn 55 LITI EE 59 Table of Contents xiii xiv Table of Contents List of Figures 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 3 1 3 2 3 3 3 4 3 5 4 2 4 3 5 1 5 2 5 3 5 4 5 5 5 6 Waters 2996 PDA Detector Dimensions iene 2 Detector Rear Panel io ttu otto e beoe naain 3 Example of IEEE 488 Cable Connections ie 4 Locating the IEEE 488 Switches us ie i te dd 5 Analog Out Terminal SP irrita reali 7 Event Input Output Terminal Strip i 8 Compression Screw Assembly ANERER 11 2996 Detector Indicator Lights degkeet Eeer 12 Removing the Flow Cell Assembly eee 21 Flow Cell and Fluidic Connections Assemblies 22 Disassembling the Plow Cell osos 23 Lamp Power Connector and Mounting Screws ees 26 pd WM MEC ccm 27 Optics Assembly Light Path at da 30 Benzene Spectrum at 1 2
7. available tighten the screws to 16 in oz 0 113 N m STOP Attention Be careful not to overtighten the screws Check for leaks If you find any leaks repeat step 5 7 Repeat steps 1 through 5 to reassemble the other side of the flow cell 3 1 4 Installing the Flow Cell Assembly Attention The alignment of the flow cell in the optics bench is critical to detector operation Be careful not to damage the flow cell body To install the flow cell assembly 1 While you hold the flow cell assembly in a vertical orientation Figure 3 2 insert the assembly into the optics bench Note that the flow cell is self aligning and uses the guide pins on the optics bench 2 Gently push the front of the assembly until it seats on the front alignment pins Hand tighten the thumbscrews 4 Reconnect the fluidic lines 24 Maintenance 5 Replace the front cover 6 Flush the flow cell Section 3 1 1 Flushing the Flow Cell 3 2 Replacing the Lamp Replace the lamp in the 2996 Detector when either of the following conditions exists e The sampling rate requires an excessively long exposure time more than 100 milliseconds e Intensity is low enough that sensitivity is not sufficient for your method Note An improperly inserted flow cell could appear to be a problem with the lamp Caution To avoid electrical hazards and exposure to UV light turn off the power and disconnect the power cord before you begin this procedure
8. detector waste outlet Solvent in drain line Leak from flow cell gasket Rebuild flow cell with a new gasket Section 3 1 3 Disassembling and Cleaning the Flow Cell Leak from flow cell fittings Check fittings for overtightening or undertightening and replace fittings if necessary 2 2 User Initiated Diagnostics Note The system administrator can restrict access to the 2996 Detector diagnostics by disabling user access to Run Samples For details refer to the Millennium Help There are two types of user initiated PDA diagnostic tests Internal Tests Tests run by the instrument firmware that help you determine the source of a malfunction These tests do not require connections to external devices Interactive Tests Tests that check analog output and event input output signal communications between the detector and connected external devices These tests require connections to pump flow and or test equipment You can run all user initiated diagnostics from Run Samples in the Millennium software For more information on Run Samples and PDA diagnostics refer to the M illennium Help User Initiated Diagnostics 17 2 3 PDA Calibration You can adjust or calibrate the 2996 Detector to ensure that wavelength readings are accurate Recalibrate the 2996 Detector only if the Wavelength Accuracy diagnostic in the Internal Diagnostics tests fails Calibration allows you to correct sma
9. element As concentration increases the chemical and instrumental requirements of Beer s law may be violated resulting in a deviation from absorbance versus concentration linearity Figure 4 4 The absorbance of mobile phase can reduce the linear range by the amounts shown in Appendix C Mobile Phase Absorbance Absorbance Working Range Background Absorbance Concentration Figure 4 4 Absorbance as a Function of Concentration 36 Principles of the 2996 PDA Detector Optics Dark Current Photodiodes lose charge over time even when they are not exposed to light The amount of charge lost is called dark current At the start of a chromatographic run the 2996 Detector closes the shutter to take a dark current reading for each diode The shutter closes after the exposure time is calculated and stays closed for the same interval as the exposure time The detector subtracts the dark current values from the current values recorded during absorbance measurements for both the sample and the reference spectra Reference Spectrum Immediately after the dark current measurement and before any components are eluted the 2996 Detector records a reference spectrum The reference spectrum is a measure of lamp intensity and mobile phase absorbance over the interval specified in the exposure time taken with the shutter open Note For best results the reference spectrum should be representative of the initial mobile phase Note
10. nm Resolution eee 32 Photodiodes Discharged by Light esee 33 Absorbance as a Function of Concentration i 36 Comparing Spectra of Two CompoundS ie 42 Plotting Vectors for Two Spectra sees 43 Spectra with a Large Spectral Contrast Angle sess 45 Spectra with a Small Spectral Contrast Angle sess 46 Absorbance Spectra of a Compound at Two Concentrations 47 Effects of pH and Solvent Concentration on the Absorbance Spectrum of DEAMIRO BENZ EE 49 List of Figures xv xvi List of Figures List of Tables 1 1 1 2 1 3 1 4 A 1 B 1 C 1 Sit ReguirementS EE 1 Event In Terminal Specifications on TTL or Switch Closure 9 Event Out Terminal Specifications on Contact Closure 9 LED Indicator Sequences During Startup esses 12 2996 Detector Troubleshooting dese deed 15 Optics Assembly Components eese sienne entente entente 30 2096 Detector SpectEcatloriSa cartacea lello gita 51 Spate RE 53 Mobile Phase Absorbance Measured Against Air or Water 55 List of Tables xvii xviii List of Tables Preface The Waters 2996 PDA Detector Operator s Guide describes the procedures for installing maintaining and troubleshooting the Waters 2996 PDA Detector It also describes detector optics and the principles of Spect
11. ou temp rature lev e Vorsicht bebe Oberfl che oder hohe Temperatur Precauzione superficie calda o elevata temperatura Precauci n superficie caliente o temperatura elevada Commonly Used Symbols Continued Caution risk of electric shock high voltage Attention risque de commotion lectrique haute tension Vorsicht Elektroschockgefahr Hochspannung Precauzione rischio di scossa elettrica alta tensione Precaucion peligro de descarga el ctrica alta tensi6n Caution risk of needle stick puncture Attention risques de perforation de la taille d une aiguille Vorsicht Gefahr einer Spritzenpunktierung Precauzione rischio di puntura con ago Precauci n riesgo de punci n con aguja Caution ultraviolet light Attention rayonnement ultrviolet Vorsicht Ultraviolettes Licht Precauzione luce ultravioletta Precauci n emisiones de luz ultravioleta 2996 PDA Detector Information Intended Use The Waters 2996 PDA Detector can be used for in vitro diagnostic testing to analyze many compounds including diagnostic indicators and therapeutically monitored compounds When you develop methods follow the Protocol for the Adoption of Analytical Methods in the Clinical Chemistry Laboratory American Journal of Medical Technology 44 1 pages 30 37 1978 This protocol covers good operating procedures and techniques necessary to validate system and method performance Biological Ha
12. the fuse block below the power cord plug Figure 3 5 on the rear panel Squeeze Side Clips to Access Fuses Figure 3 5 Fuse Block Replacing the Fuses 27 Squeeze the two side clips on the fuse block while you pull out the block Remove the fuses from the block then install the new fuses Position the fuse block with the small tab pointing down then push in the block until the side clips engage 6 Connect the power cord then power on the 2996 Detector 28 Maintenance Chapter 4 Principles of the 2996 PDA Detector Optics To use the Millennium PDA software effectively you must be familiar with the principles of operation of the optics and electronics of the Waters 2996 PDA Detector 4 1 2996 Detector Optics The 2996 Detector is an ultraviolet visible light UV Vis spectrophotometer with 512 photodiodes Optical resolution of 1 2 nm per diode Operating wavelength range from 190 to 800 nm The light path through the optics assembly of the 2996 Detector is shown in Figure 4 1 2996 Detector Optics 29 Photodiode Array Grating 50 um Aperture Shutter Beamsplitter Assembly Assembly Reference Diode Spectrographic Flow Cell Lamp and Mirror and Mask Assembly Lamp Optics Figure 4 1 Optics Assembly Light Path Table 4 1 describes the optics assembly components in the 2996 Detector Table 4 1 Optics Assembly Components Component Function Lamp and lamp Focuses light from the deuter
13. values can improve signal to noise ratios Computing Absorbance Data Points The 2996 Detector calculates absorbance values before transmitting the data to the Millennium database To calculate absorbance the 2996 Detector Computes the absorbance at each diode using the dark current and reference spectrum Section 4 4 1 Calculating Absorbance Averages the absorbances at a particular wavelength as specified in the spectra per second sample rate and reports the average as a single data point Section 4 4 2 Resolution e Can apply a filter Section 4 4 3 Filtering Data Calculating Absorbance The detector computes absorbance by subtracting the dark current and reference spectrum from the acquired spectrum Absorbance is based on the principles of Beer s Law Computing Absorbance Data Points 35 Beer s Law The relationship between the quantity of light of a particular wavelength arriving at the photodiode and the concentration of the sample passing through the flow cell is described by the Beer Lambert Law commonly called Beer s Law Beer s Law is expressed as A elc where A absorbance e molar absorptivity I path length 1 0 cm in the 2996 Detector normal flow cell c molar concentration 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
14. 01462 Figure 3 1 Removing the Flow Cell Assembly 6 Use the Phillips screwdriver to loosen the three thumbscrews that hold the flow cell assembly to the optics bench and the thumbscrew that secures the bracket holding the fluidic connections then detach the bracket 7 Pull the flow cell assembly gently toward you to remove it from the detector Figure 3 2 Flow Cell Maintenance 21 Flow Cell Body Lens Holder Assembly Da 4 a Fluidic O Bracket Connections O TP01463 Figure 3 2 Flow Cell and Fluidic Connections Assemblies 3 1 3 Disassembling and Cleaning the Flow Cell STOP Attention The lens surface finish and the alignment of the lenses are critical to the performance of the 2996 Detector Be careful not to touch or damage the lenses and the lens holders Attention To prevent lens contamination use powder free gloves when disassembling inspecting cleaning or replacing parts within the flow cell or when removing or replacing the flow cell within ts assembly Required Materials e TORX M T10 screwdriver e Small flat blade screwdriver Lens tissue or nonparticulating swab 22 Maintenance HPLC grade methanol Belleville spring washer Flow cell gasket e Powder free gloves Procedure To disassemble and clean the flow cell and lenses 1 Use the TORX T10 screwdriver to remove the three screws that secure one of the lens holder assemblies Figure 3 3 Slot for Removi
15. 24 in 61 cm Height 8 5 in 22 cm Bench support Capable of supporting 31 5 pounds 14 3 kg Clearance At least 4 in 10 cm on the back side for ventilation Power Grounded ac 100 to 240 Vac Installation Site Requirements 1 4 11 5 in 29 cm E Sample Inlet Sample Outlet Drain Line Figure 1 1 Waters 2996 PDA Detector Dimensions 1 2 Power Connections Ensure that power connections for the 2996 PDA Detector are made according to the procedures that follow Operating Voltage The 2996 PDA Detector has a universal input power supply that requires no voltage adjustment The electrical power requirements for the Waters 2996 PDA Detector are Voltage range 100 to 240 Vac Total power 100 VA Frequency range 50 to 60 Hz Fuses The 2996 PDA Detector is shipped with fuses rated for North American operation If you operate the 2996 PDA Detector in another location install the IEC rated fuses supplied in 2 Installation the Waters 2996 Detector Startup Kit in the fuse holder in the rear of the detector refer to Section 3 3 Replacing the Fuses Connecting the Power Cord Connect one end of the 2996 Detector power cord to the rear panel power receptacle Figure 1 2 and the other end to a power outlet AC Input TP01452 Figure 1 2 Detector Rear Panel 1 3 Millennium Workstation Connections The 2996 Detecto
16. 67 0 40 0 10 0 01 1M Sodium citrate 2 48 2 84 2 31 2 02 1 49 0 54 0 12 0 03 0 02 0 01 10 mM Sodium formate 1 00 0 73 0 53 0 33 0 20 0 03 lt 0 01 10 mM Sodium 1 99 0 75 0 19 0 06 0 02 0 01 0 01 0 01 0 01 lt 0 01 phosphate 100 mM pH 6 8 Tris HCl 20mM 1 40 0 77 0 28 0 10 0 04 lt 0 01 pH 7 0 Tris HCI 20mM 1 80 1 90 1 11 0 43 0 13 lt 0 01 pH 8 0 Mobile Phase Absorbance 57 Table C 1 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 1 vial L 0 67 0 29 0 13 0 05 0 03 0 02 0 02 0 02 0 02 lt 0 01 PIC B6 1 vial L 2 46 2 50 2 42 2 25 1 83 0 63 0 07 lt 0 01 PIC B6 low UV 0 01 lt 0 01 1 vial L PIC D4 1 vial L 0 03 0 03 0 03 0 03 0 02 0 02 0 02 0 02 0 02 0 01 Detergents BRIJ 35 1 0 06 0 03 0 02 0 02 0 02 0 01 lt 0 01 CHAPS 0 1 2 40 2 32 1 48 0 80 0 40 0 08 0 04 0 02 0 02 0 01 SDS 0 1 0 02 0 01 lt 0 01 Triton 2 48 2 50 2 43 2 42 2 37 2 37 0 50 0 25 0 67 1 42 X 100 0 1 Tween M 20 0 21 0 14 0 11 0 10 0 09 0 06 0 05 0 04 0 04 0 03 0 1 58 Mobile Phase Absorbance Index A Absorbance maximum 48 mobile phase 55 photometric error 48 solvent change effects 49 Waters 29
17. 96 calculations 35 37 Acquisition Auto Exposure parameter 34 Exposure Time parameter 34 Analog output specifications 6 7 Aperture width 31 Auto Exposure parameter 34 B Beer s law 36 48 C Calibration 18 Column connecting 10 Compression fittings 10 Connections column 10 events 8 fluidic 10 non IEEE 488 6 rear panel 3 terminal strip 8 Contact closures 6 Contacting Waters Technical Service 15 17 Conventions documentation xx D Dark current 37 Data acquisition Auto Exposure parameter 34 Exposure Time parameter 34 Derived vectors 43 44 Diagnostics 17 Documentation conventions xx E Electrical connections 2 Events connections 6 8 electrical specifications 9 terminal strip connections 7 Exposure Time parameter 34 F Fittings 10 Flow cell cleaning 23 exploded view 23 flushing 20 maintenance 19 removing 20 Fluid connecting lines 10 fittings 10 Fuses IEC rated 2 maintenance 27 replacement 27 Index 59 I IEC rated fuses 2 Inputs 6 8 9 Installation electrical 2 fluidic 10 site selection 1 Instrument method Auto Exposure parameter 34 Exposure Time parameter 34 L Lamp hardware theory 30 replacement 25 26 M Maintenance flow cell 19 fuse 27 lamp 25 PDA detector 19 28 Match Angle photometric error effects 48 Maximum absorbance 48 Millennium Chromatography Manager connections 3 Mobile phase absorbances 55 wavelengths 55 N Noise effects 47 Nonidealit
18. Caution The lamp and housing are extremely hot To avoid the possibility of contacting hot surfaces allow the lamp to cool for 15 minutes before you handle the lamp assembly or surfaces close to the lamp Note If lamp intensity is low but the lamp has not been used for 2000 hours you may be able to increase lamp intensity by cleaning the flow cell Section 3 1 Flow Cell Maintenance Absorbance by the mobile phase also affects the apparent lamp intensity For example acetonitrile is more transparent than methanol at wavelengths less than 220 nm Attention Do not touch the lamp glass while unpacking or inserting the lamp Touching lamp glass damages the lamp and reduces life expectancy Attention To prevent lamp glass contamination use powder free gloves when removing or replacing the lamp Caution The lamp may be very hot Please allow it to cool for at least 15 minutes before you attempt to remove it Replacing the Lamp 25 Required Materials e Slotted head screwdriver Powder free gloves Caution To avoid electrical hazards when you perform the following procedure power off the 2996 Detector and disconnect the power cord Procedure To replace the lamp 1 Power off the 2996 Detector disconnect the power cord and allow the lamp to cool for at least 15 minutes Caution To avoid the possibility of contacting hot surfaces wait at least VAN 15 minutes after powering off the detector before you handle the lamp
19. EE 488 Communication Conpecttong eee 6 1 4 1 Connecting Analog Output Cables AAA 6 1 4 2 Connecting Event Cables rotos 7 1 BEudie Connections E 9 1 6 Starting Up and Shutting Down the Detector eene 11 Chapter 2 Diagnostics and Calibration se 15 2S MEE EVI LC O EE E EE R 15 22 User Initiated Diagnostic cia AUS itus Pati tdv eie EAR 17 2 37 PDA Calibrations eeu eode t iet 18 Chapter 3 Maintenance T RO ii 19 3A Flow Cell Maintenance vespa leda lele e RE auge 19 3 11 Flushing the Plow Gell ipe bande Qe Eon inue ne pavit 20 3 1 2 Removing the Flow Cell odo sola pci illa ib 20 3 1 3 Disassembling and Cleaning the Flow Cell 22 3 1 4 Installing the Flow Cell Assembly eee 24 Table of Contents Xi 3 2 Replacinp the Lamp EE 25 3 3 Replacing the uge eoi ei tei eoi Le teri e eria erect E A 27 Chapter 4 Principles of the 2996 PDA Detector Optics 29 Al 2990 Detector ls A A E R S 29 42 o De A 31 4 3 Measuring Light at the Photodiode iia 32 4 4 Computing Absorbance Data Points i 35 4 4 1 Calculating Absorbance s coe toe eta e te edet e 35 AAD Resolution carrara dirottati 38 445 Filtering EE 38 Chapter 5 Spectral Contrast Theory 41 5 1 Comparing Absorbance Spectra nete tette ride 41 5 2 Representing Spectra as NEES eee kee een 42 5 2 1
20. For extremely long exposure times the dark current and reference spectrum readings may take several seconds to finish Absorbance The 2996 Detector calculates the absorbance for each diode at the end of each exposure time using the following equation Absorbanceg log Kal Rn Dn where S obtained during sample analysis D obtained during the dark current test R obtained from the reference spectrum n diode number Computing Absorbance Data Points 37 4 4 2 Resolution The data reported by the 2996 Detector to the Millennium database can be the average of a number of data points After calculating absorbance the detector averages absorbance values based on Spectral resolution Sample rate Averaging Spectral Data Based on Resolution Spectral resolution or bandwidth is the wavelength interval in nanometers between data points in an acquired spectrum The minimal resolution of the 2996 Detector is 1 2 nm For example in 3D mode the 2996 Detector averages three adjacent diodes for each reported wavelength when the spectral resolution is set in the Millennium software to 3 6 nm In 2D mode absorbance values are computed based on the bandwidth setting 2D mode is supported in Millennium software versions 4 0 or higher Averaging Chromatographic Data Based On Sample Rate Sample rate is the number of data points per second reported to the Millennium database The number of times the photodiodes are
21. Spectral Contrast technique Representing Spectra as Vectors 43 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 technique forms a vector for each spectrum in an n dimensional space The two spectral vectors are compared mathematically to compute the angle between the two vectors Just as in 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 5 3 Spectral Contrast Angles Spectra that have the same shape have vectors that point in the same direction Spectra that have different shapes have vectors that point in different directions The angle between the two vectors of any two spectra the Spectral Contrast angle quantifies the magnitude of the shape difference between the spectra The Spectral Contrast angle is 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 near 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 s
22. Waters 2996 PDA Detector Operator s Guide Waters 34 Maple Street Milford MA 01757 71500023202 Revision C NOTICE 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 the use of this document 2001 WATERS CORPORATION PRINTED IN THE UNITED STATES OF AMERICA ALL RIGHTS RESERVED THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER Millennium PIC and Waters are registered trademarks and busLAC E and PowerStation are trademarks of Waters Corporation Micromass is a registered trademark and MassLynx is a trademark of Micromass Ltd All other trademarks or registered trademarks are the sole property of their respective owners UKAS QUALITY MANAGEMENT Note When you use the instrument follow generally accepted procedures for quality control and methods development If you observe a change in the retention of a particular compound in the resolution between two compounds or in peak shape immediately determine the reason for the changes Until you determine the cause of a
23. ample STOP Attention To avoid damaging the detector flow cell do not touch the flow cell window Cautions Cautions provide information essential to the safety of the operator For example Caution To avoid possible burns turn off the lamp at least 15 minutes before removing it for replacement or adjustment and unplug the power cord before performing maintenance procedures Caution To avoid chemical or electrical hazards always observe safe laboratory practices when operating the system d Caution To avoid possible electrical shock and injury always turn off the detector XXi xxii Chapter 1 Installation 1 1 The Waters 2996 Photodiode Array PDA Detector operates in any standard laboratory environment The detector requires electrical power sample and waste fluidic lines and either the Millennium or MassLynx workstations Optional connections on the detector rear panel allow communication with chart recorders data integrators and other instruments that are not compatible with Millennium or MassLynx software control Installation Site Requirements Install the 2996 PDA Detector at a site that meets the specifications in Table 1 1 and Figure 1 1 Table 1 1 Site Requirements Factor Specification Ambient temperature 4 to 40 C 39 to 104 F not to vary more than 1 C per hour to prevent drift Relative humidity 20 to 80 noncondensing Bench space Width 11 5 in 29 cm Depth
24. ample in the flow cell The array consists of 512 photodiodes arranged in a row Each photodiode acts as a capacitor by holding a fixed amount of charge Light striking a photodiode discharges the diode Figure 4 3 The magnitude of the discharge depends on the amount of light striking the photodiode 32 Principles of the 2996 PDA Detector Optics Deuterium Sample in flow cell Lu Lamp absorbs at specific wavelengths Grating ZE T Cell Light from grating dispersed into 1 2 nm wavelength beams continuously discharges diodes Mirror Figure 4 3 Photodiodes Discharged by Light The 2996 Detector measures the amount of current required to recharge each photodiode The current is proportional to the amount of light transmitted through the flow cell over the interval specified by the diode exposure time Exposure Time The 2996 Detector recharges each diode and reads the recharging current one diode at a time The interval between two readings of an individual diode is the exposure time The 2996 Detector requires less than 10 msec to sequentially read all of the diodes in the array and process the data The minimum exposure time is 10 msec You can set exposure time from 10 to 500 msec For example if an exposure time is set to 50 milliseconds the 2996 Detector 1 Recharges diode 1 and reads the current required to recharge diode 1 2 Recharges diode 2 and reads the current required to recharge diode 2 Measu
25. ay Detector flow cell lamp and fuse Caution To avoid possible electric shock do not remove the 2996 Detector power supply covers The power supply does not contain user serviceable components 3 1 Flow Cell Maintenance The flow cell requires maintenance when e The reference spectrum changes The cell fluid leaks out of the drain tube e The Lamp diagnostic in the Millennium PDA Diagnostics window fails and the lamp status light is on Table 2 1 The 2996 Detector causes high backpressure Note Conditions other than a dirty flow cell may cause decreased lamp intensity For more information refer to Chapter 2 Diagnostics and Calibration Flow cell maintenance consists of Flushing the flow cell Removing the flow cell Disassembling and cleaning the flow cell e Installing the flow cell assembly Flow Cell Maintenance 19 3 1 1 Flushing the Flow Cell Required Materials HPLC grade water HPLC grade methanol If the flow cell requires cleaning first try flushing it with solvent Procedure To flush the flow cell 1 Selecta solvent compatible with the samples and mobile phases that you have been using If you have been using buffers flush with HPLC grade water for 10 minutes at 1 mL min then switch to a low surface tension solvent such as methanol STOP Attention Ensure that the solvent is miscible with the previous mobile phase 2 Set pump flow to 1 mL min then run the pump for 10 minut
26. bance ratios of multiple wavelength pairs Comparing Absorbance Spectra 41 Compound A 245 nm Compound B 257 nm Compound A CR UNE MENU ERE DEC E RECEN fat L 240 00 Zo ah a dh KAAL nm Sd 5063 nm 0 4595 AU Figure 5 1 Comparing Spectra of Two Compounds 5 2 Representing Spectra as Vectors The Spectral Contrast technique uses vectors to quantify differences in the shapes of spectra Spectral Contrast converts baseline corrected spectra to vectors and then compares the vectors Spectral vectors have two properties 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 AU across the collected wavelength range Vector direction contributes to the identification of a compound since the direction is a function of the absorbance spectrum of the compound 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 vector derived from the 2996 PDA Detector can include absorbances in any range from 190 to 800 nm To enhance spectral sensitivity set the bench resolution to 1 2 nm 42 Spectral Contrast Theory 5 2 1 Vectors Derived from Two Wavelengths
27. change do not rely on the separation results Note The Installation Category Overvoltage Category for this instrument is Level II The Level Il Category pertains to equipment that receives its electrical power from a local level such as an electrical wall outlet Attention Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment Important Toute modification sur cette unit n ayant pas t express ment approuv e par l autorit responsable de la conformit la r glementation peut annuler le droit de l utilisateur exploiter l quipement Achtung Jedwede nderungen oder Modifikationen an dem Ger t ohne die ausdr ckliche Genehmigung der f r die ordnungsgem e Funktionst chtigkeit verantwortlichen Personen kann zum Entzug der Bedienungsbefugnis des Systems f hren Avvertenza eventuali modifiche o alterazioni apportate a questa unit e non espressamente approvate da un ente responsabile per la conformit annulleranno l autorit dell utente ad operare l apparecchiatura Atenci n cualquier cambio o modificaci n realizado a esta unidad que no haya sido expresamente aprobado por la parte responsable del cumplimiento puede anular la autorizaci n de la que goza el usuario para utilizar el equipo Caution Use caution when working with any polymer tubing under pressure e Always wear eye protection when near pressur
28. d by statistical and thermal variations can be predicted from the instrument noise in the baseline region of a chromatogram Undesirable Effects 47 5 4 2 Photometric Error At high absorbances generally greater than 1 AU a combination of effects can produce slight departures about 1 from Beer s Law due to photometric error Although photometric errors at this level may have a negligible effect on quantitation they can 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 Mobile Phase Absorbance Note For more information about the effects of the photometric error curve refer to Principles of Instrumental Analysis third edition by Douglas A Skoog Saunders College Publishing 1985 pp 168 172 5 4 3 Solvent Changes As long as solvent concentration and composition do not change isocratic operation the background absorbance if any by the solvent remains constant A change however in solvent pH or composition such as occurs in gradient operation can affect the intrinsic spectral shape of a compound as shown in Figure 5 6 5 4 4 Threshold Angle 48 In add
29. d of an analog signal cable into the positive and negative terminals of Analog Out 1 Figure 1 5 Tighten the two screws to secure the and wires 6 Installation 3 Connect the other end of the analog signal cable to the appropriate analog input terminal on the external device being sure to maintain negative to negative and positive to positive continuity 4 Reinstall the Analog Output Terminal strip Removable Analog Output 2 Terminal Strip Ri ii i nalog Ou IEE 488 ADDRESS Analog Out 2 TP01456 Figure 1 5 Analog Out Terminal Strip 1 4 2 Connecting Event Cables The 2996 Detector has four terminal strip connections for contact closure signals e Two input signal terminals generally used for inject start Two output programmable event table signal terminals If an inject start signal is not available over the IEEE 488 bus you must provide a signal at Event In 1 terminal on the 2996 Detector rear panel Manual injectors such as the Rheodyne 77251 provide a cable that connects the injector to an Event In terminal on the 2996 Detector rear panel The values of the event output signals generated by the 2996 Detector are specified by parameter values set from the Millennium Workstation For details refer to the Millennium Help Non IEEE 488 Communication Connections 7 Required Materials Small flat blade screwdriver One electrical insulation stripping tool Eve
30. ects of a 0 1 to 3 second RC filter Computing Absorbance Data Points 39 40 Principles of the 2996 PDA Detector Optics Chapter 5 Spectral Contrast Theory 5 1 This chapter explains the theory behind the Spectral Contrast technique which is used to compare UV Vis absorbance spectra collected by the 2996 Detector Spectral Contrast makes use of the fact that different compounds have differently shaped absorbance spectra This chapter describes how Spectral Contrast represents absorbance spectra as vectors When applied to the UV Vis absorbance data collected by the 2996 Detector the Spectral Contrast technique determines whether differences between spectra are due to the presence of multiple compounds in the same peaks coelution or due to nonideal conditions such as noise photometric error or solvent effects Comparing Absorbance Spectra The shape of an absorbance spectrum is determined by the relative absorbance at different wavelengths The shape of a compound s absorbance spectrum is a characteristic of that compound at the solvent and pH conditions under which the absorbance spectrum is measured Figure 5 1 shows the absorbance spectra for the two compounds A and B The ratio of the absorbance at 245 nm to the absorbance at 257 nm is approximately 2 2 for compound A and 0 7 for compound B The absorbance ratios of two wavelength pairs is a limited spectral comparison For more information you need to compare the absor
31. es 3 Test the lamp energy by performing the Lamp diagnostic test For details refer to the Millennium Help If the lamp diagnostic fails and the lamp has not been used more than 2000 hours or 1 year whichever comes first disassemble the flow cell and clean the flow cell components using the procedure described in Section 3 1 2 Removing the Flow Cell 3 1 2 Removing the Flow Cell Note You do not need to shut down the 2996 Detector to remove and replace the flow cell Required Materials 5 16 inch open end wrench Phillips screwdriver Powder free gloves 20 Maintenance Procedure To remove the 2996 Detector flow cell 1 Setthe flow to 0 0 mL min 2 Poweroff the solvent delivery system or pump to avoid exposure to chemicals the inlet or outlet fluidic lines while there is pressure in the chromatographic i Caution To avoid the possibility of leaking mobile phase do not disconnect system Always vent your system before disconnecting fluidic lines 3 Use the 5 16 inch wrench to disconnect the fluidic lines at the front of the detector Lift up the 2996 detector front cover and pull the front cover from the detector chassis 5 Open the flow cell access door by pulling the black thumbtab then pull the door gently toward you Figure 3 1 Thumbscrews Holding Flow Cell Assembly and Bracket Fluidic Connections O Inside Bracket TP
32. g Outputs The 2996 Detector provides two unattenuated 1 volt per absorbance unit analog output channels to integrators chart recorders or other components Event Inputs and Outputs The 2996 Detector sends and receives contact closure signals to and from other instruments You make all non IEEE 488 communication cable connections to the 2996 Detector at the rear panel on the analog event terminal Figure 1 2 disturbances that may affect the performance of this instrument do not use cables longer than 9 8 feet 3 meters when you make connections to the screw type barrier terminal strips In addition ensure that you always connect the shield of each cable to chassis ground STOP Attention To meet the regulatory requirements of immunity from external electrical 1 4 1 Connecting Analog Output Cables The values of the analog output signals generated by the 2996 Detector are specified by parameter values set from the Millennium Workstation For details refer to the Millennium Help Required Materials e One small flat blade screwdriver One electrical insulation stripping tool Analog signal cables included with the Waters 2996 Detector Startup Kit Procedure To connect the 2996 Detector to a device that receives analog output signals 1 Pull off the analog out terminal strip from the 2996 Detector rear panel Figure 1 5 This step simplifies the following steps 2 Insert the appropriate bare wires at one en
33. g boxes Also includes reference information and procedures for performing Xix all tasks required to use the Millennium software Included as part of the Millennium software Millennium Software Getting Started Guide Provides an introduction to the Millennium System Describes the basics of how to use Millennium software to acquire data develop a processing method review results and print a report Also covers basics for managing projects and configuring systems Millennium PDA Software Getting Started Guide Describes the basics of how to use Millennium PDA software to develop a PDA processing method and to review PDA results Millennium System Installation and Configuration Guide Describes Millennium software installation including the stand alone workstation PowerStation system and client server system Discusses how to configure the computer and chromatographic instruments as part of the Millennium system Also covers the installation configuration and use of acquisition servers such as the LAC E module the busLAC E card and interface cards used to communicate with serial instruments Waters 2996 PDA Detector Qualification Workbook Describes qualification procedures for the 2996 PDA Detector Documentation Conventions The following conventions may be used in this guide Convention Usage Bold Bold indicates user action such as keys to press menu selections and commands For example C
34. ies 47 Non IEEE 488 connections 6 60 Index O Outputs 6 8 9 P Parts spare 53 Photodiode array 32 Photometric error 48 Power connections 2 Purity Angle photometric error effects 48 R Rear panel connections 3 Reference spectrum 37 S Shutdown procedure 13 Solvent Angle photometric error effects 48 Solvent changes 48 Spare parts 53 Specifications analog output 6 7 event inputs 9 event outputs 9 Waters 2996 51 Spectra derived vectors 43 44 spectral shape differences 47 vectors 42 Spectral Contrast derived vectors 43 44 spectral shape differences 47 theory 41 49 vectors 42 Spectral resolution 31 Spectrum match spectral shape differences 47 Start up procedure 11 T Terminal strip connections 7 8 diagram 8 Threshold angle 47 Troubleshooting 15 18 Tubing cutting 10 U Undesirable effects shape differences 47 V Vectors derived from multiple wavelengths 44 derived from two wavelengths 43 spectra representing 42 spectral contrast 42 W Waters 2996 absorbance calculations 35 37 aperture width 31 dark current 37 detector optics overview 29 31 hardware theory 29 39 photodiode array overview 32 reference spectrum 37 spare parts 53 specifications 51 spectral resolution 31 Waters Technical Service contacting 15 17 Wavelength accuracy 18 derived vectors 43 44 mobile phase absorbances 55 Index 61 Index 62
35. inform que si le mat riel est utilis d une facon 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 unter Umst nden nicht ordnungsgem funktionieren Precauzione l utente deve essere al corrente del fatto che se l apparecchiatura viene usta in un modo specificato dal produttore la protezione fornita dall apparecchiatura potr essere invalidata Advertencia El usuario deber saber que si el equipo se utiliza de forma distinta a la especificada por el fabricante las medidas de protecci n del equipo podr an ser insuficientes Caution To protect against fire hazard replace fuses with those of the same type and rating Attention Remplacez toujours les fusibles par d autres du m me type et de la m me puissance afin d viter tout risque d incendie Vorsicht Zum Schutz gegen Feuergefahr die Sicherungen nur mit Sicherungen des gleichen Typs und Nennwertes ersetzen Precauzione per una buona protezione contro i rischi di incendio sostituire i fusibili con altri dello stesso tipo e amperaggio Precauci n sustituya los fusibles por otros del mismo tipo y caracter sticas para evitar el riesgo de incendio Caution To avoid possible electrical shock power off the instrument and disconnect the power cord before servicing the instrument Attention Afin d v
36. iter toute possibilit de commotion lectrique mettez hors tension l instrument et d branchez le cordon d alimentation de la prise avant d effectuer la maintenance de l instrument Vorsicht Zur Vermeidung von Stromschl gen sollte das Ger t vor der Wartung abgeschaltet und vom Netz getrennt werden Precauzione per evitare il rischio di scossa elettrica spegnere lo strumento e scollegare il cavo di alimentazione prima di svolgere la manutenzione dello strumento Precauci n para evitar choques el ctricos apague el instrumento y desenchufe el cable de alimentaci n antes de realizar cualquier reparaci n en el instrumento Commonly Used Symbols Direct current Courant continu Gleichstrom Corrente continua Corriente continua Alternating current Courant alternatif Wechselstrom Corrente alternata Corriente alterna Protective conductor terminal Borne du conducteur de protection Schutzleiteranschluss Terminale di conduttore con protezione Borne del conductor de tierra Frame or chassis terminal Borne du cadre ou du chassis Rahmen oder Chassisanschluss Terminale di struttura o telaio Borne de la estructura o del chasis Caution or refer to manual Attention ou reportez vous au guide Vorsicht oder lesen Sie das Handbuch Prestare attenzione o fare riferimento alla guida Act e con precauci n o consulte la guia Caution hot surface or high temperature Attention surface chaude
37. ition to computing Spectral Contrast angles the Spectral Contrast technique also computes a Threshold angle The Threshold angle is the maximum Spectral Contrast angle between spectra that can be attributed to nonideal phenomena Comparison of a Spectral Contrast angle to its Threshold angle can assist in determining if the shape difference between spectra is genuine that is generated by mixtures that are dissimilar In general a Spectral Contrast angle less than its Threshold angle indicates that shape differences can be attributed to nonideal phenomena alone and that there is no evidence for genuine differences between the spectra A Spectral Contrast angle greater than its Threshold angle indicates that the shape differences are due to genuine differences between the spectra When automating the spectra contrast comparison the maximum absorbance of the spectra must not exceed 1 AU Spectral Contrast Theory Absorbance Absorbance Effect of pH Wavelength nm Effect of Concentration Note position of Maxima can be shifted Wavelength nm Figure 5 6 Effects of pH and Solvent Concentration on the Absorbance Spectrum of p Aminobenzoic Acid Undesirable Effects 49 50 Spectral Contrast Theory Appendix A Detector Specifications Table A 1 lists the 2996 PDA Detector specifications Table A 1 2996 Detector Specifications Item Specification Dimensions Width 11 5 in 29 cm Depth 24 in 61 cm
38. ium source lamp via a mirror through a optics beamsplitter to the flow cell Beamsplitter and Reflects part of the light back to a reference diode which measures reference diode the intensity of the light emitted by the lamp The detector uses this measurement to keep the lamp output constant Flow cell Houses the segment of the flow path containing eluent and sample assembly through which the polychromatic light beam passes This arrange ment of optical components with the flow cell positioned between the lamp and the grating is commonly called reversed optics 30 Principles of the 2996 PDA Detector Optics Table 4 1 Optics Assembly Components Continued Component Function Spectrograph The mirror focuses light transmitted through the flow cell onto the mirror and mask aperture at the entrance to the spectrographic portion of the optics The mirror mask defines the beam of light focused on the spec trograph mirror Aperture Controls wavelength resolution and intensity of light striking the photodiodes The width of the aperture is 50 um Shutter assembly Prevents light from reaching the photodiode array except during sampling and calibration For details on the dark current see Section 4 4 1 Calculating Absorbance Grating Disperses the light into bands of wavelengths and focuses those wavelength bands onto the plane of the photodiode array Second order Reduces the contribution of second order reflection of UV
39. ized polymer tubing e Extinguish all nearby flames e Do not use Tefzel tubing that has been severely stressed or kinked e Do not use Tefzel tubing with tetrahydrofuran THF or concentrated nitric or sulfuric acids e Be aware that methylene chloride and dimethyl sulfoxide cause Tefzel tubing to swell which greatly reduces the rupture pressure of the tubing Attention soyez tr s prudent en travaillant avec des tuyaux de polym res sous pression e Portez toujours des lunettes de protection quand vous vous trouvez a proximit de tuyaux de polym res e Eteignez toutes les flammes se trouvant a proximit e N utilisez pas de tuyau de Tefzel fortement ab m ou d form e N utilisez pas de tuyau de Tefzel avec de l acide sulfurique ou nitrique ou du t trahydrofurane THT e Sachez que le chlorure de m thyl ne et le sulfoxyde de dim thyle peuvent provoquer le gonflement des tuyaux de Tefzel diminuant ainsi fortement leur pression de rupture Vorsicht Bei der Arbeit mit Polymerschl uchen unter Druck ist besondere Vorsicht angebracht e In der N he von unter Druck stehenden Polymerschl uchen stets Schutzbrille tragen e Alle offenen Flammen in der Nahe l schen e Keine Tefzel Schl uche verwenden die stark geknickt oder berbeansprucht sind e Tefzel Schl uche nicht f r Tetrahydrofuran THF oder konzentrierte Salpeter oder Schwefels ure verwenden e Durch Methylenchlorid und Dimethylsulfoxid k nnen Tef
40. k 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 compression fitting Figure 1 7 at both ends of the column outlet line and at one end of the detector outlet line Installation Compression Screw Ferrule End Must be Straight Tubing and Smooth to Prevent DR Dead Volume Distance Determined by the Union or Column Fitting TP01139 Figure 1 7 Compression Screw Assembly 4 Seat one end of the column outlet tubing in the fitting of the column outlet then tighten the compression screw about 3 4 turn past finger tight using the 5 16 inch open end wrench 5 Seat the other end of the tubing in the fitting of the detector inlet then tighten the compression screw as in step 4 6 Seat the end of the detector outlet tubing with the compression fitting in the detector outlet fitting then tighten the compression screw about 3 4 turn past finger tight Insert the other end of the tubing in the waste container Attention To avoid damage to the flow cell avoid pressures approaching its maximum allowable pressure 1000 psi 70 kg cm 1 6 Starting Up and Shutting Down the Detector The entire startup procedure takes less than 1 minute Once completed you should allow the 2996 Detector to warm up for at least 1 hour before running an analysis Follow the procedures in this section to ensure reliable detector performance S
41. l can cause shutter diagnostic to fail To prevent air bubbles from forming check that there is 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 lamp Section 3 1 2 Removing the Flow Cell Shutter failure Shutter failed Run the Shutter diagnostic For details message see Millennium Help Detector not responding to Millennium Workstation Detector not connected to busLAC E or to LAC E Acquisition Server in the Millennium Workstation Check IEEE 488 cable connections tighten connectors Incorrect IEEE 488 address Ensure that the 2996 Detector IEEE 488 address is unique and within the range 2 to 29 see the Millennium System Installation and Configuration Guide 2 Rescan the IEEE 488 bus For details see Millennium Help Diagnostics and Calibration Table 2 1 2996 Detector Troubleshooting Continued Symptom Possible Cause Corrective Action Change in reference spectrum Mobile phase contains gas or is contaminated Prepare fresh mobile phase and degas thoroughly Air bubbles trapped in flow cell Reseat and check alignment of flow cell Flush the flow cell or apply slight backpressure on the detector waste outlet To prevent air bubbles check that there is a 1 to 2 foot 30 to 60 cm length of 0 009 inch 0 23 mm ID tubing connected to the
42. lick Next to go to the next page Italic Italic indicates information that you supply such as variables It also indicates emphasis and document titles For example Replace file_name with the actual name of your file Courier Courier indicates examples of source code and system output For Courier Bold example The SVRMGR gt prompt appears Courier bold indicates characters that you type or keys you press in examples of source code For example At the LSNRCTL gt prompt enter set password oracle to access Oracle XX Convention Usage Keys The word key refers to a computer key on the keypad or keyboard Screen keys refer to the keys on the instrument located immediately below the screen For example The A B screen key on the 2414 Detector displays the selected channel Three periods indicate that more of the same type of item can optionally follow For example You can store filename 1 filename2 in each folder gt A right arrow between menu options indicates you should choose each option in sequence For example Select File gt Exit means you should select File from the menu bar then select Exit from the File menu Notes Notes call out information that is helpful to the operator For example Note Record your result before you proceed to the next step Attentions Attentions provide information about preventing possible damage to the system or equipment For ex
43. light filter less than 370 nm to the light intensity observed at visible wave lengths greater than 370 nm Photodiode array An array of 512 photodiodes arranged linearly The diode width and spacing provide a single wavelength resolution of 1 2 nm 4 2 Resolving Spectral Data The ability to distinguish similar spectra depends on photodiode spacing and the bandwidth of the light striking the photodiode The bandwidth of the light striking the photodiodes depends on the aperture width The aperture width determines Attainable wavelength bandwidth at the photodiode array Intensity of the light reaching the photodiode array optical throughput The aperture creates a narrow beam that reflects from the grating to the photodiode array The wavelength that strikes a particular diode depends on the angle of reflection from the grating Figure 4 2 shows an absorbance spectrum of benzene obtained from the 2996 Detector using the 50 um aperture In this spectrum the wavelength resolution is sufficient to resolve five principal benzene absorption peaks Resolving Spectral Data 31 2191 min Absorbance rae ft fa et remore E EN 22000 230 00 24000 250 00 26000 270 00 28000 290 00 nm Figure 4 2 Benzene Spectrum at 1 2 nm Resolution 4 3 Measuring Light at the Photodiode The 2996 Photodiode Array Detector measures the amount of light striking the photodiode array to determine the absorbance of the s
44. ll errors in wavelength which may be caused by aging optics or excessive vibration You calibrate the 2996 Detector using the PDA Calibration window which you access from Run Samples and which allows you to View the effects of exposure time on photodiode saturation for a given wavelength range Verify the wavelength location of the deuterium spectrum Balmer lines 486 0 nm and 656 1 nm Recalibrate to set the 486 nm peak at the proper wavelength Ensure precise data for library matching STOP Attention Recalibrating the wavelength requires that spectral libraries be reentered Note The system administrator can restrict access to the PDA Calibration window by disabling access to Run Samples Note Ensure that the flow cell is clean before you check calibration Section 3 1 1 Flushing the Flow Cell To prepare for calibration 1 Setthe pumpto deliver 1 mL min of degassed methanol for 10 minutes If methanol is not miscible with the previous solvent flush with a miscible sovent before switching to methanol 2 If you have been using buffers flush with HPLC quality water at 1 mL min for 10 minutes then switch to methanol for 10 minutes Note Ensure that the solvent is miscible with the previous mobile phase For information on performing calibration refer to the Millennium Help 18 Diagnostics and Calibration Chapter 3 Maintenance This chapter covers maintenance of the Waters 2996 Photodiode Arr
45. ment in your chromatographic system up to a maximum of 14 IEEE 488 instruments Note Keep in mind cable length limitations when you set up your system For a list of IEEE 488 interface guidelines refer to the Millennium System Installation and Configuration Guide 4 Installation 4 Ensure that all IEEE 488 cable screws are fastened finger tight Cable Lengths The maximum length of all cables connecting instruments to one busLAC E is 2 meters multiplied by the number of devices or 20 meters whichever is smaller The maximum length of cable between devices is 4 meters Note The maximum number of devices to be connected together is 14 1 3 2 Setting the IEEE 488 Address To set the IEEE 488 address for the 2996 Detector Use a small screwdriver or similar device to set the IEEE 488 switches on the detector rear panel Figure 1 4 to the IEEE 488 address of the 2996 Detector The address must be a number from 2 to 29 and must be different than that of any other component connected to your acquisition server Refer to the Millennium System Installation and Configuration Guide for the correct IEEE 488 GPIB switch settings IEEE 488 Cable Connection IEEE 488 Address Switches TP01457 Figure 1 4 Locating the IEEE 488 Switches Millennium Workstation Connections 5 1 4 Non IEEE 488 Communication Connections Non IEEE 488 communication connections on the 2996 Detector include Analo
46. nces During Startup Continued Lamp LED Status LED Indicates Troubleshooting OFF FLASHING The 2996 is running power on self tests FLASHING FLASHING 2996 has failed one of Contact Waters Technical the self tests Service OFF FLASHING The 2996 is running Confidence tests ON FLASHING Lamp start was successful The 2996 is starting calibration ON FLASHING for more The 2996 may not Could be air bubble in flow than 30 seconds have calibrated cell Table 2 1 Contact correctly Waters Technical Service ON ON Calibration was successful Shutting Down the Detector To shut down the 2996 Detector 1 Ifthe mobile phase contains buffers set the solvent delivery system or pump to deliver 1 mL min of HPLC grade water for 10 minutes Otherwise set the solvent delivery system or pump to deliver 1 mL min of degassed methanol for 10 minutes 2 Press the 0 1 Off On switch on the front panel of the detector to the 0 Off position Starting Up and Shutting Down the Detector 13 14 Installation Chapter 2 Diagnostics and Calibration 2 1 The Waters 2996 Photodiode Array Detector automatically runs a series of internal diagnostics upon start up The indicator LEDs on the front of the detector and messages at the Millennium Workstation show the results of the start up internal diagnostics Figure 1 8 If you need to determine the cause of a problem during operation of the detector you can run the same in
47. ng Flow Cell Disk Lens Assembly NSOE l Lens Assembly Flow Cell A Sa TO Spring Washer Screws Lens Assembly 4 4 O A Ze Gasket Belleville Spring Washer Flow Cell Disk TP01464 Figure 3 3 Disassembling the Flow Cell 2 Use the small flat blade screwdriver to gently pry the lens assembly from the flow cell body at the slots Attention Solvents other than methanol may damage a disassembled flow cell In normal use the gasket protects the lens holder from solvents 3 Use a lens tissue or a nonparticulating swab to wipe the lens with methanol Flow Cell Maintenance 23 4 Remove and discard the gasket 5 Repeat steps 1 through 4 to remove disassemble and clean the other lens holder assembly 6 Use methanol and a nonparticulating swab to clean the flow cell body Reassembling the Flow Cell To reassemble the flow cell Figure 3 3 1 Insert a replacement gasket into one side of the flow cell body 2 Align the screw holes of the lens assembly with the holes in the flow cell body 3 Place the new Belleville spring washers with the concave side facing out onto the lens assembly 4 Place the flow cell disk over the lens assembly 5 Insert the three screws using the TORX T10 driver to gradually tighten each screw alternating between the screws in a clockwise pattern Tighten until the screws meet the flow cell disk then tighten each screw 1 4 turn If a torque screwdriver is
48. nt signal cables included in the Waters 2996 Detector Startup Kit Procedure To connect the 2996 Detector to an external event input or output device Pull off the event terminal strip from the rear panel Figure 1 6 This simplifies the following steps l Insert the bare wires at one end of the event signal cable into the positive and negative slots of the appropriate event input or output terminal Figure 1 6 Tighten the two screws to secure the and wires 2 Connect the other end of the event signal cable to the appropriate event input or event output terminal on the external device 3 Reinstall the event terminal strip Removable Event Input Output Terminal Strip Event IEE Ate ADDRESS QOQQOQOQOQOQOQ00 TP01455 Figure 1 6 Event Input Output Terminal Strip 8 Installation Electrical Specifications Before you connect an external device to an event input or output terminal refer to the electrical specifications in Table 1 2 and Table 1 3 Table 1 2 Event In Terminal Specifications on TTL or Switch Closure Parameter Specification Low trigger lt 1 8 V High trigger gt 3 0 V Protected to 30 V Minimum pulse width 30 msec this may limit compatibility with valves that require a quick pulse Maximum current 5mA Table 1 3 Event Out Terminal Specifications on Contact Closure Parameter Specification Maximum power 10 W Maximum current 0 5 A at 20 V Ma
49. otometric error high sample concentration or variations in solvent conditions The spectra in Figure 5 5 for example show how instrument noise can affect the shape of an absorbance spectrum of one compound This effect is most likely to occur at low concentrations where the signal to noise ratio 1s low Note that the Spectral Contrast angle between these absorbance spectra of the same compound is 3 4 46 Spectral Contrast Theory 5 4 5 4 1 Normalized Spectra of a Compound at Different Concentrations Spectral Contrast Angle 3 4 Normalized Absorbance Wavelength nm Figure 5 5 Absorbance Spectra of a Compound at Two Concentrations 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 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 Section 5 4 4 Detector Noise Statistical and thermal variations add electrical noise to the absorbance measurements made by the 2996 Detector The noise manifests itself as fluctuations in the baseline known as baseline noise The magnitude of any absorbance differences cause
50. pectra do not overlap at any wavelength To illustrate the relationship between Spectral Contrast angle and spectral shape differences consider the pairs of spectra shown in Figure 5 3 Figure 5 4 and Figure 5 5 Spectra with Different Shapes In Figure 5 3 the absorbance spectra of two compounds A and B are distinctly different and therefore have a large Spectral Contrast angle 44 Spectral Contrast Theory Spectral Contrast Angle 62 3 Compound A 0 e 3 Compound B 6 o Q lt o 0 N o E o z T T T T T T T T T T T T T T T T T T T T 220 00 24000 260 00 220 00 300 00 320 00 340 00 Wavelength nm Figure 5 3 Spectra with a Large Spectral Contrast Angle Spectra with Similar Shapes In Figure 5 4 the absorbance spectra of two compounds A and B are similar and therefore have a small Spectral Contrast angle 3 09 Spectral Contrast Angles 45 Spectral Contrast Angle 3 0 Compound A Compound B Normalized Absorbance 22000 240 00 260 00 29000 30000 320 00 340 00 Wavelength nm Figure 5 4 Spectra with a Small Spectral Contrast Angle Differences Between Spectra of the Same Compound Small but significant differences between absorbance spectra can occur because of factors other than those due to the absorbance properties of different compounds For example multiple spectra of the same compound may exhibit slight differences because of detector noise ph
51. r requires signal connections to the Millennium Workstation over the IEEE 488 bus All detector control and data acquisition communications take place over the IEEE 488 bus Note If an inject start signal is not available over the IEEE 488 bus you must provide a signal at the Event In 1 terminals on the 2996 Detector rear panel Section 1 4 2 Connecting Event Cables Millennium Workstation Connections 3 1 3 1 Connecting the IEEE 488 Cable To connect the 2996 Detector to a Millennium Workstation 1 Connect one end of the IEEE 488 cable to the IEEE 488 receptacle on the rear panel of the 2996 Detector Connect the other end of the cable stackable connector for daisy chaining additional instruments to the IEEE 488 connector on any of the other instruments in your chromatographic system Figure 1 3 L1 Waters Millennium IEEE 488 busLAC E Card Cable on Workstation IEEE 488 a IEEE 488 Cable Connector H dq Waters Waters 2996 Alliance Separations Module Detector TP01544 Figure 1 3 Example of IEEE 488 Cable Connections Note The order in which you connect IEEE 488 devices to the busLAC E card on the workstation is not important For example you can connect the 2690 Separations Module before or after the 2996 Detector 2 Use another IEEE 488 cable to connect to the stackable connector on the first instrument and the IEEE 488 connector on another instrument 3 Repeat step 2 for each IEEE 488 instru
52. ral Contrast used in the Millennium software for analyzing the data from the PDA detector Also included is information on vector analysis mobile phase absorbance specifications This guide is intended for individuals who need to install operate maintain and troubleshoot the 2996 PDA Detector It is also intended for users who need to understand the Spectral Contrast principles underlying the processing of PDA Detector data by Millennium software Organization This guide contains the following Chapter 1 describes how to install and set up the 2996 Detector Chapter 2 describes how to troubleshoot the 2996 Detector Chapter 3 describes how to replace the flow cell the lamp and the fuse Chapter 4 explains the principles involved in resolving spectral data measuring light at the photodiode verifying wavelengths and computing absorbance data Chapter 5 describes the calculations used for Spectral Contrast Appendix A provides the specifications of the 2996 PDA Detector Appendix B lists recommended spare parts Appendix C provides a table of absorbances at several wavelengths for common mobile phases Related Documentation Waters Licenses Warranties and Support Provides software license and warranty information describes training and extended support and tells how Waters handles shipments damages claims and returns 32 TE Millennium Help Describes all Millennium windows menus menu selections and dialo
53. re than 90 Provides proper settings for signal to noise and dynamic range for each run The Auto Exposure time setting may not support certain combinations of sampling rates wavelength ranges or filter time constant settings required for your analysis If this is the case you can set the exposure time manually to adjust the exposure time from experiment to experiment Using the Exposure Time Parameter The Exposure Time parameter enables you to manually set the length of time the photodiodes are exposed to light before they are read The supported range is 10 to 500 msec 34 Principles of the 2996 PDA Detector Optics Note Changing exposure times within a set of samples can cause changes in baseline noise Be aware that increasing the Exposure Time parameter has the potential to saturate the photodiodes A longer exposure time may cause the 2996 Detector to lose the signal at certain wavelengths because of diode saturation When specifying the Exposure Time select a value that provides settings for an optimum signal to noise ratio over the wavelength range of your analysis see the next topic Optimizing the Signal to Noise Ratio Optimizing the Signal to Noise Ratio 4 4 4 4 1 To optimize signal to noise ratios choose an acquisition wavelength range that includes only the wavelengths of interest and over which the mobile phase absorbs minimally Appendix C Mobile Phase Absorbance Setting the bandwidth to higher
54. read during the sample rate interval is dependent on the exposure time For example if exposure time is 25 msec and sample rate is 1 sec then readings per data point are 1000 msec 4 25 msec S The readings are averaged and reported as a single data point Combining Spectral Resolution and Sample Rate Spectral resolution and sample rate have opposite effects on noise and spectral detail Increasing the value of the spectral resolution parameter and decreasing the number of spectra per second decrease the size of the data file Note The data storage rate is based on wavelength range spectral resolution and sample rate which are set in the General tab of the 2996 PDA Instrument Method Editor For details refer to the Millennium Help 4 4 3 Filtering Data Use the General tab of the 2996 PDA Instrument Method Editor for details refer to the Millennium Help to apply an optional noise filter the Filter Response parameter to 38 Principles of the 2996 PDA Detector Optics the data sent to the Millennium software database A noise filter of 1 second is the default value which provides a good signal to noise ratio for most chromatographic separations Note the following with regard to filtering data e The noise filter is a digital low pass filter e The filter calculates a data point that is a modified rolling average for a wavelength over a number of readings The filter values are comparable to the eff
55. ring Light at the Photodiode 33 3 Sequentially recharges and reads the current required to recharge all the remaining 510 photodiodes 4 Waits approximately 45 msec before beginning the recharge and reading sequence with diode 1 after all diodes have been recharged and read You set the exposure time parameter in the General tab of the 2996 PDA Instrument Method Editor You can specify either Auto Exposure or Exposure Time For details refer to the Millennium Help Note For best signal to noise performance adjust the wavelength range optimize autoexposure computations For details refer to the Millennium Help Using the Auto Exposure Parameter The Auto Exposure time parameter allows the 2996 Detector optics to calculate the optimum exposure time needed to recharge the diodes based on lamp energy lamp spectrum mobile phase absorbance and the chosen wavelength range using a single Deuterium light source from 190 to 800 nm To minimize detector noise Auto Exposure adjusts the exposure time from 50 to 90 of full scale The Auto Exposure time setting ensures that the photodiodes are Notsaturating due to overexposure Operating above the range of normal dark current discharge With auto exposure enabled the 2996 Detector Calculates exposure time at the start of a run based on maximum light intensity within the selected wavelength range Limits the exposure so that no diode within the given wavelength range discharges mo
56. tarting Up the Detector To start up the 2996 Detector 1 In your instrument method set the solvent delivery system or pump to deliver 1 mL min of degassed mobile phase For details refer to the Millennium Help Starting Up and Shutting Down the Detector 11 Note 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 2 Flush the detector for 10 minutes or until no bubbles appear in the outlet line Press the 0 1 Off On switch on the front panel of the detector Figure 1 8 to the 1 On position 4 Observe the Lamp and Status indicator LEDs on the front panel of the detector Figure 1 8 If both lights remain illuminated the detector passed the internal diagnostics If either indicator light blinks or is off refer to the troubleshooting tables in Chapter 2 Diagnostics and Calibration 5 Wait 1 hour for the 2996 Detector to stabilize before acquiring data WATERS 2996 Photodiode Array Detector On Off Switch Status Indicator Lamp Indicator TP01460 Figure 1 8 2996 Detector Indicator Lights Table 1 4 LED Indicator Sequences During Startup Lamp LED Status LED Indicates Troubleshooting OFF OFF No power or CPU Check ac source and main failure fuse Contact Waters Tech nical Service 12 Installation Table 1 4 LED Indicator Seque
57. ternal diagnostics from the Millennium Workstation Additional information about the performance of the detector is also available through the PDA Calibration window accessed from Run Samples in the Millennium software If you encounter a problem that you cannot troubleshoot Section 2 1 Diagnostics contact Waters Technical Service at 800 252 4752 U S and Canadian customers only Other customers call your local Waters subsidiary or your local Waters Technical Service Representative in Milford Massachusetts U S A for assistance Diagnostics Refer to Table 2 1 to troubleshoot problems encountered during startup diagnostics and during detector operation Table 2 1 2996 Detector Troubleshooting Symptom Possible Cause Corrective Action Both LEDs off No power 1 Check line cord connections 2 Check outlet for power Blown fuse Replace fuse Section 3 3 Replacing the Fuses Status light blinks and The 2996 is running lamp light is off Confidence tests Diagnostics 15 Table 2 1 2996 Detector Troubleshooting Continued Symptom Possible Cause Corrective Action Status light blinks and Failed startup Reseat and check alignment of flow lamp light on diagnostics cell Flush the flow cell Section 3 1 1 Flushing the Flow Cell Insufficient energy Flush the flow cell Section 3 1 1 reaching photodiode Flushing the Flow Cell array because of air bubble or dirty flow cel
58. ximum voltage 24 V RMS Attention To avoid damage to the 2996 Detector electronics be sure you make the proper electrical connections as outlined in this section 1 5 Fluidic Connections Caution To avoid chemical hazards always observe safe laboratory practices when handling solvents Refer to the Material Safety Data Sheets for the solvents in use Required Materials e 5 16 inch open end wrench 0 009 inch 0 23 mm ID stainless steel tubing included in the 2996 Detector Startup Kit Fluidic Connections 9 10 Stainless steel tubing cutter or scribing file Pliers plastic covered or with cloth e Compression screw assemblies three Procedure To make fluidic connections to the 2996 Detector 1 Measure the lengths of tubing needed to connect The column outlet to the 2996 Detector inlet Note Keep the length of this tubing as short as possible to prevent band broadening The 2996 Detector outlet to a waste collection bottle Note Ensure the length of this tubing is at least 1 to 2 feet 30 to 60 cm to prevent air bubbles from forming in the flow cell Cut the two lengths of tubing as follows a Use a Waters 1 16 inch stainless steel tubing cutter or a file with a cutting edge to scribe the circumference of the tubing at the desired 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 wor
59. zard When you analyze physiological fluids take all necessary precautions and treat all specimens as potentially infectious Precautions are outlined in CDC Guidelines on Specimen Handling CDC NIH Manual 1984 Calibration Follow acceptable methods of calibration with pure standards to calibrate methods Use a minimum of five standards to generate a standard curve The concentration range should cover the entire range of quality control samples typical specimens and atypical specimens Quality Control Routinely run three quality control samples Quality control samples should represent subnormal normal and above normal levels of a compound Ensure that quality control sample results are within an acceptable range and evaluate precision from day to day and run to run Data collected when quality control samples are out of range may not be valid Do not report this data until you ensure that chromatographic system performance is acceptable General Maintenance To clean the outside of the Waters 2996 PDA Detector use only a soft lint free paper or cloth dampened with mild soap and water Table of Contents Nr xix Chapter 1 Ins eR P 1 1 1 Installation Site Requirements vecs eae oet lets din 1 1 2 Power Connections un onere erat td 2 1 3 Millennium Workstation Connections ee 3 1 3 1 Connecting the IEEE 488 Cable acini itt op iot 4 1 3 2 Setting the IEEE 488 Address colera ile 5 1 4 Non IE
60. zel Schl uche quellen dadurch wird der Berstdruck des Schlauches erheblich reduziert Precauzione prestare attenzione durante le operazioni con i tubi di polimero sotto pressione e Indossare sempre occhiali da lavoro protettivi nei pressi di tubi di polimero pressurizzati e Estinguere ogni fonte di ignizione circostante e Non utilizzare tubi Tefzel soggetti a sollecitazioni eccessive o incurvati e Non utilizzare tubi Tefzel contenenti tetraidrofurano THF o acido solforico o nitrico concentrato e Tenere presente che il cloruro di metilene e il dimetilsolfossido provocano rigonfiamento nei tubi Tefzel che riducono notevolmente il limite di pressione di rottura dei tubi stessi Advertencia manipular con precaucion los tubos de polimero bajo presion e Protegerse siempre los ojos a proximidad de tubos de polimero bajo presion e Apagar todas las llamas que est n a proximidad e No utilizar tubos Tefzel que hayan sufrido tensiones extremas o hayan sido doblados e No utilizar tubos Tefzel con tetrahidrofurano o acidos nitrico o sulfurico concentrados e No olvidar que el cloruro de metileno y el xido de azufre dimetilo inflan los tubos Tefzel lo que reduce en gran medida la presi n de ruptura de los tubos A Caution The user 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

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