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5-1

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

Содержание 2998

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

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

Страница 4: ...in this section Applicable symbols Audience and purpose This guide is intended for personnel who install operate and maintain the Waters 2998 Photodiode Array PDA detector Symbol Definition Manufactur...

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

Страница 6: ...vi EC authorized representative Waters Corporation Micromass UK Ltd Floats Road Wythenshawe Manchester M23 9LZ United Kingdom Telephone 44 161 946 2400 Fax 44 161 946 2480 Contact Quality manager...

Страница 7: ...ity control v ISM classification v ISM Classification ISM Group 1 Class B v EC authorized representative vi 1 2998 PDA Detector Optics Principles 1 1 Detector optics 1 2 Calculating absorbance 1 3 Flo...

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

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

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

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

Страница 12: ...xii Table of Contents...

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Страница 61: ...that the new fuses are properly rated for your requirements and then insert them into the holder and the holder into the power entry module gently pushing until the assembly locks into position 6 Reco...

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Страница 83: ...des also include the hazard symbols with accompanying text statements describing the hazards and telling you how to avoid them This appendix presents all the safety symbols and statements that apply t...

Страница 84: ...electric shocks ultraviolet radiation exposures and others When the following symbols appear in a manual s narratives or procedures their accompanying text identifies the specific risk and explains ho...

Страница 85: ...ing Risk of explosion Warning Risk of eye injury Warning Pressurized nonmetallic or polymer tubing can burst Observe these precautions when working around such tubing Wear eye protection Extinguish al...

Страница 86: ...Ensure that the nitrogen supply pressure never falls below 690 kPa 6 9 bar 100 psi during an analysis in which flammable solvents are used Also ensure a gas fail connection is connected to the LC syst...

Страница 87: ...nisms and other biological materials To avoid infection with these agents assume that all biological fluids are infectious observe Good Laboratory Practices and consult your organization s biohazard s...

Страница 88: ...ler le droit de l utilisateur exploiter l quipement Achtung Jedwede nderungen oder Modifikationen an dem Ger t ohne die ausdr ckliche Genehmigung der f r die ordnungsgem e Funktionst chtigkeit verantw...

Страница 89: ...e trouvant proximit de l instrument Evitez d utiliser des tubes s v rement d form s ou endommag s Evitez d utiliser des tubes non m talliques avec du t trahydrofurane THF ou de l acide sulfurique ou n...

Страница 90: ...iamenti nei tubi non metallici riducendo notevolmente la pressione di rottura dei tubi stessi Advertencia se recomienda precauci n cuando se trabaje con tubos de pol mero sometidos a presi n El usuari...

Страница 91: ...Warnings that apply to all Waters instruments A 9 Nonmetallic Tetrahydrofuran THF Methylene chloride Dimethyl sulfoxide THF...

Страница 92: ...tueuses Vorsicht Der Benutzer wird darauf aufmerksam gemacht dass bei unsachgem er Verwenddung des Ger tes die eingebauten Sicherheitseinrichtungen unter Umst nden nicht ordnungsgem funktionieren Atte...

Страница 93: ...l instrument Vorsicht Zum Schutz gegen Feuer die Sicherungen nur mit Sicherungen ersetzen deren Typ und Nennwert auf den Tafeln neben den Sicherungsabdeckungen des Ger ts gedruckt sind Attenzione per...

Страница 94: ...e can appear in instrument user manuals and on the instrument s front or rear panels Electrical power on Electrical power off Standby Direct current Alternating current Protective conductor terminal F...

Страница 95: ...ng symbols A 13 Handling symbols These handling symbols and their associated text can appear on labels affixed to the outer packaging of Waters instrument and component shipments Keep upright Keep dry...

Страница 96: ...A 14 Safety Advisories...

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

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

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

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

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

Страница 102: ...s generally maintain their purity from lot to lot use them to ensure the best possible results Preparation checklist The following solvent preparation guidelines help to ensure stable baselines and go...

Страница 103: ...nol temperature affects solvent miscibility If you are running a high temperature application consider the effect of the higher temperature on solvent solubility buffers dissolved in water can precipi...

Страница 104: ...17 7 15 4 4 Methyl acetate 0 45 56 3 15 17 260 4 5 Methyl ethyl ketone 0 43 80 0 17 330 4 5 Cyclohexanone 2 24 155 7 28 210 4 5 Nitrobenzene 2 03 210 8 14 20 4 6 Benzonitrile 1 22 191 1 15 19 4 8 Diox...

Страница 105: ...e solvents receive a dual M number The first number always lower than 16 indicates the degree of miscibility with highly lipophilic solvents The second number applies to the opposite end of the scale...

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

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

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

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

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

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

Страница 112: ...Acetic acid 1 230 Sodium chloride 1 M 207 Ammonium acetate 10 mM 205 Sodium citrate 10 mM 225 Ammonium bicarbonate 10 mM 190 Sodium dodecyl sulfate 190 BRIJ 35 0 1 190 Sodium formate 10 mM 200 CHAPS...

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

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

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

Страница 116: ...1 2 33 2 42 2 50 2 45 2 37 1 96 0 50 0 12 0 04 0 01 Buffers and Salts Ammonium acetate 10 mM 1 88 0 94 0 53 0 29 0 15 0 02 0 01 Ammonium bicarbonate 10 mM 0 41 0 10 0 01 0 01 EDTA disodium 1 mM 0 11 0...

Страница 117: ...um citrate 10 mM 2 48 2 84 2 31 2 02 1 49 0 54 0 12 0 03 0 02 0 01 Sodium formate 10 mM 1 00 0 73 0 53 0 33 0 20 0 03 0 01 Sodium phosphate 100 mM pH 6 8 1 99 0 75 0 19 0 06 0 02 0 01 0 01 0 01 0 01 0...

Страница 118: ...02 0 02 0 02 0 02 0 02 0 01 Detergents BRIJ 35 1 0 06 0 03 0 02 0 02 0 02 0 01 0 01 CHAPS 0 1 2 40 2 32 1 48 0 80 0 40 0 08 0 04 0 02 0 02 0 01 SDS 0 1 0 02 0 01 0 01 Triton X 100 0 1 2 48 2 50 2 43...

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

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

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

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

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