Waters 2998 Overview And Maintenance Manual
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Waters 2998 Overview And Maintenance Manual

Photodiode array detector
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2998 Photodiode Array Detector
Overview and Maintenance Guide
715004753
Copyright © Waters Corporation
2015 – 2017
Revision B
All rights reserved

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Summary of Contents for Waters 2998

  • Page 1 2998 Photodiode Array Detector Overview and Maintenance Guide 715004753 Copyright © Waters Corporation 2015 – 2017 Revision B All rights reserved...
  • Page 2 December 5, 2017, 715004753 Rev. B Page ii...

  • Page 3: Trademarks

    This document is believed to be complete and accurate at the time of publication. In no event shall Waters Corporation be liable for incidental or consequential damages in connection with, or arising from, its use. For the most recent revision of this document, consult the Waters website (waters.com).

  • Page 4: Contacting Waters

    Contacting Waters Contact Waters with enhancement requests or technical questions regarding the use, transportation, removal, or disposal of any Waters product. You can reach us via the Internet, telephone, or conventional mail. Waters contact information Contacting medium Information Internet The Waters Web site includes contact information for Waters locations worldwide.

  • Page 5: Fcc Radiation Emissions Notice

    FCC radiation emissions notice Changes or modifications not expressly approved by the party responsible for compliance, could void the user's authority to operate the equipment. This device complies with Part 15 of the FCC Rules. Operation is subject to the following two conditions: (1) this device may not cause harmful interference, and (2) this device must accept any interference received, including interference that may cause undesired operation.

  • Page 6: Intended Use Of The 2998 Photodiode Array Detector

    Serial number Part number catalog number Audience and purpose Tis guide is intended for personnel who install, operate, and maintain the Waters 2998 Photodiode Array (PDA) detector. Intended use of the 2998 Photodiode Array Detector Waters designed the 2998 PDA detector to analyze and monitor various types of compounds. The 2998 PDA detector is not intended for use with in vitro diagnostic applications.

  • Page 7: Emc Considerations

    Class B products are suitable for use in both commercial and residential locations and can be directly connected to a low voltage, power-supply network. EC authorized representative Waters Corporation Stamford Avenue Altrincham Road Wilmslow SK9 4AX UK...
  • Page 8 December 5, 2017, 715004753 Rev. B Page viii...

  • Page 9: Table Of Contents

    Customer comments ........................iii Contacting Waters ........................iv Safety considerations ........................ iv Safety hazard symbol notice..................iv Considerations specific to the 2998 Photodiode Array Detector ......... iv FCC radiation emissions notice ..................v Electrical power safety notice ..................v Equipment misuse notice....................v Safety advisories ......................
  • Page 10 2.7 Plumbing the 2998 PDA detector ....................38 2.7.1 Connecting the detector to the gas supply ..............39 2.8 Starting up and shutting down the 2998 PDA detector .............. 40 2.8.1 Starting the detector....................... 40 2.8.2 Monitoring LEDs ......................41 2.8.3 Shutting down the detector ....................
  • Page 11 A.2 Notices ............................71 A.3 Bottles Prohibited symbol ......................71 A.4 Required protection ........................71 A.5 Warnings that apply to all Waters instruments and devices ............72 A.6 Warnings that address the replacing of fuses ................75 December 5, 2017, 715004753 Rev. B...
  • Page 12 A.7 Electrical and handling symbols ....................76 A.7.1 Electrical symbols......................76 A.7.2 Handling symbols ......................77 B Specifications ....................... 79 B.1 Physical specifications ......................79 B.2 Environmental specifications ..................... 79 B.3 Electrical specifications ......................79 B.4 Performance specifications ....................... 80 B.5 Optical component specifications .....................
  • Page 13 C.9.3 Wavelength selection for chromophore detection ............91 C.9.4 Mobile phase absorbance ..................... 92 December 5, 2017, 715004753 Rev. B Page xiii...
  • Page 14 December 5, 2017, 715004753 Rev. B Page xiv...

  • Page 15: 2998 Pda Detector Optics Principles

    2998 PDA Detector Optics Principles To use the 2998 Photodiode Array (PDA) detector effectively, you must understand the principles that underlie operation of the detector’s optics and electronics. Detector optics The detector is an ultraviolet/visible light (UV/Vis) spectrophotometer. With a photodiode array of 512 photodiodes and an optical resolution of 1.2 nm/pixel, the detector operates within a range of...

  • Page 16: Calculating Absorbance

    Table 1–1: Optics assembly components Component Function Lamp Deuterium source lamp. M1 mirror Focuses light from the deuterium source lamp. Window Used to help minimize air infiltration into the lamp housing. Filter flag/shutter Flag positions for measuring open (sample) and blocked (dark) beam energies and a third for wavelength verification.

  • Page 17: Flow Cell Operating Principles

    The Waters TaperSlit flow cell, so-called because of the shape of the flow cell exit face, matches the shape of the spectrograph slit. Compared to a conventional flow cell with a cylindrical shape, the PDA detector achieves higher light throughput for a given spectral resolution with the TaperSlit cell design.

  • Page 18: Resolving Spectral Data

    Resolving spectral data Together with photodiode spacing, the detector’s 50-µm 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 a result, similar spectra are more effectively distinguished. The grating images the slit onto the photodiode array.

  • Page 19: Optimizing The Signal-To-Noise Ratio

    Figure 1–5: Photodiodes discharged by light Sample in flow cell absorbs at specific Deuterium lamp wavelengths. Grating Flow cell Light from grating dispersed onto diodes. Slit Mirror The detector measures the amount of charge required to recharge each photodiode. The charge is proportional to the amount of light transmitted through the flow cell over the interval specified by the diode exposure time.

  • Page 20: Filtering Data

    Figure 1–6: Example of how baseline noise increases with higher sampling rates 5 Hz 10 Hz 20 Hz 40 Hz 1.4.3 Filtering data On the General tab of the PDA Instrument Method Editor you can apply an optional noise filter to the data acquired.
  • Page 21 The following table lists the digital filter settings for the allowable data rates: Table 1–2: Digital filter settings for data rates Sampling Slow Normal Fast rate 4.000 2.000 1.000 2.000 1.000 0.500 0.800 0.400 0.200 0.400 0.200 0.100 0.200 0.100 0.050 0.100 0.050...

  • Page 22: Computing Absorbance Data Points

    Figure 1–7: Filter-time constant comparison Time (minutes) Although the peak shape shows some distortion and the signal output is delayed with different Tip: filter-time constants, the peak area remains the same. Computing absorbance data points The absorbance data points are calculated by the detector and then transmitted to the database (MassLynx or Empower software).

  • Page 23: Dark Current

    1.5.1 Dark current Photodiodes discharge even when they are not exposed to light. This discharge is called dark current. The dark current is updated by closing the shutter to take a dark current reading for each diode. After updating, the detector opens the shutter and then subtracts the dark current values as shown in the above equation.
  • Page 24 Figure 1–8: Resolution comparison for anthracene 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 252 nm at 1.2 nm, height 0.53 252 nm at 6.0 nm, height 0.45 252 nm at 12.0 nm, height 0.35 1.5.3.2 Average chromatographic sampling rate...

  • Page 25: Reference Wavelength Compensation

    1.5.4 Reference wavelength compensation A compensated reference wavelength collects wide-band absorbance data in a region of the spectra where no known analytes exist. It is used to reduce detector drift and wander, which can affect integration quality. The detector calculates the compensation value by averaging the absorbance values within a range of wavelengths that you select.
  • Page 26 December 5, 2017, 715004753 Rev. B Page 26...

  • Page 27: Setting Up The Detector

    The chapter provides the information necessary to set up the 2998 PDA detector. Before you begin To install the 2998 PDA detector, you must know how, in general, to set up and Requirement: operate laboratory instruments and computer-controlled devices and also how to handle solvents.

  • Page 28: Selecting A Laboratory Site

    Ensure that the instrument serial number on the rear panel nameplate or inside the front door corresponds to the number on the instrument validation certificate. Selecting a laboratory site To ensure the detector’s reliable operation, observe these precautions: • Do not situate it under a heating or cooling vent. •...

  • Page 29: Connecting To The Electricity Source

    Repeats steps 1 and 2 for the remaining system modules. Connecting to the electricity source The 2998 PDA detector requires a separate, grounded electricity source. The ground connection in the electrical outlet must be common and connected near the system.

  • Page 30: Connecting Signal Cables

    Connecting signal cables See also: Waters Ethernet Instrument Getting Started Guide. The following figure shows the rear panel location of the connectors used to operate the 2998 PDA detector with external devices. Figure 2–3: Location of connectors on 2998 PDA detector rear panel...
  • Page 31 Figure 2–4: 2998 PDA detector rear panel analog-out/event-in connectors Connector B (outputs) Connector A (inputs)   Analog 1 Inject Start   Analog 1 Inject Start Ground Ground   Analog 2 Lamp On/Off   Analog 2 Lamp On/Off ...

  • Page 32: Connecting The Ethernet Cable

    (LAN). At the acquisition computer, the instrument network card provides the interface that makes communication possible. You must install the Waters instrument software driver on the acquisition computer so that the computer can control the instrument. See the software installation instructions that accompany the instrument control software.

  • Page 33: Network Installation Guidelines

    2.6.2 Network installation guidelines Configurations for multiple Waters instruments use a dedicated LAN, which requires a design based on the following guidelines: • Ethernet cable • A maximum distance of 100 meters (328 feet) You must use a network switch for multiple Ethernet instruments. Network hubs are Requirement: not supported.

  • Page 34: Connecting To Other Devices

    2.6.2.1 Making inject-start signal connections When you are using an Ethernet data system with the 2998 PDA detector, the data system or controller must receive an inject-start signal from manual injector, to initiate the data-collection and time-based programs. The following table summarizes the inject-start connections for different system configurations.
  • Page 35 The firmware defaults to Autozero on injection. Tip: Table 2–4: 2998 PDA detector connections to an Alliance separations module Alliance separations module (connector B) 2998 PDA detector (connector A) Pin 1 inject start  (red) Pin 1 inject start (red) Pin 2 inject start (black)
  • Page 36 Connecting to an Empower or MassLynx data system via an eSAT/IN module To send an integrator analog output signal (–0.1 to 2.1 V) from the 2998 PDA detector to an Empower or MassLynx system (via a two-channel eSAT/IN module), connect cables shown in the following table and figure.
  • Page 37 The firmware defaults to auto zero on injection. Tip: Table 2–7: Inject-start connections to an injector (pulse duration 0 to 10 seconds) 2998 PDA detector (connector A) Injector connector Pin 1, inject start  (red) Terminal connectors Pin 2, inject start – (black) December 5, 2017, 715004753 Rev.

  • Page 38: Plumbing The 2998 Pda Detector

    Plumbing the 2998 PDA detector ® If you are setting up an ACQUITY Arc or ACQUITY Arc Bio system, you can Exception: disregard this section. Waters will connect all necessary plumbing. “Minimum bend radius for tubing” on page See also: Warning: •...

  • Page 39: Connecting The Detector To The Gas Supply

    Cut the two lengths of tubing as follows: 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.

  • Page 40: Starting Up And Shutting Down The 2998 Pda Detector

    The nitrogen source can require an adapter to fit the plastic tubing. Tip: Connect the free end to the nitrogen fitting on the rear panel of the detector. Starting up and shutting down the 2998 PDA detector Warning: • Always observe Good Laboratory Practices when you use this equipment and when you work with solvents and test solutions.

  • Page 41: Monitoring Leds

    Light-emitting diodes on the detector indicate its state of functioning. 2.8.2.1 Power LED The power LED, on the 2998 PDA detector’s front door, indicates when the instrument is powered-on or powered-off. It is steady green when the instrument is working properly. 2.8.2.2 Lamp LED The lamp LED, to the left of the power LED, indicates the lamp status.

  • Page 42: Before You Begin

    • Instrument verification and qualification • Benchtop spectrophotometer procedures 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 flow cell assembly.
  • Page 43 Figure 2–13: Location of cuvette holder Cuvette holder With the spring guide facing you, gently insert the cuvette (containing eluent) up and under the guide, with the cap facing upward (into the holder) and a frosted side of the cuvette facing upward (see the figure on page 42).
  • Page 44 December 5, 2017, 715004753 Rev. B Page 44...

  • Page 45: Maintaining The Detector

    PDA detector in good working order. Contacting Waters technical service If you are located in the USA or Canada, report malfunctions or other problems to Waters Technical Service (800 252-4752). Otherwise, phone the Waters corporate headquarters in Milford, Massachusetts (USA) or contact your local Waters subsidiary. The Waters Web site includes phone numbers and e-mail addresses for Waters locations worldwide.

  • Page 46: Spare Parts

    After disconnecting power, wait for 10 seconds before attempting to disconnect an assembly. 3.2.2 Spare parts Waters recommends that you replace only the parts mentioned in this document. For spare parts details, refer to these sources: ® •...

  • Page 47: Flushing The Flow Cell

    53). If the diagnostic test fails and the has not been used more than 2000 hours or 1 year Note: from date of purchase (whichever event occurs first), call Waters Technical Service (see page 45). 3.4.2 Replacing the flow cell Required tools and materials •...
  • Page 48 Figure 3–1: 2998 PDA detector showing the analytical flow cell Lamp Flow cell handle Flow cell Thumbscrews Remove the flow cell: • Use a 1/4-inch flat-blade screwdriver to loosen the three thumbscrews on the flow cell assembly’s front plate. •...

  • Page 49: Replacing The Lamp

    Replacing the lamp Change the lamp when it repeatedly fails to ignite or when the 2998 PDA detector fails to calibrate. The detector’s source lamp is warranted to light and pass startup diagnostic tests for 2000 hours or 1 year from the date of purchase, whichever event occurs first.
  • Page 50 Lamp gas is under slight negative pressure. To prevent shattering the Warning: glass, use care when disposing of the lamp. Waters suggests that you adequately cushion an old lamp by containing it in the packaging of its replacement before you dispose of it.

  • Page 51: Replacing The Fuses

    For continued protection against fire, replace fuses with those of the same type and rating only. The 2998 PDA detector requires two 100 to 240 Vac, 50 to 60-Hz, F 3.15-A, 250-V (fast-blow), 5 × 20 mm (IEC) fuses.
  • Page 52 Power-off the detector, and disconnect the power cord from the power-entry module. Figure 3–4: Power-entry module Power-entry module Pinch the sides of the spring-loaded fuse holder, which is below the power entry module on the rear panel of the detector. Figure 3–5: Sides of spring-loaded fuse holder Sides of fuse holder...

  • Page 53: Diagnostic Tests And Troubleshooting

    Diagnostic Tests and Troubleshooting Consult this chapter when troubleshooting problems with the 2998 PDA detector. Bear in mind, however, that the detector measures only the bulk properties of a system. Therefore, the cause of an apparent detector problem can originate with the chromatography or other system instruments.

  • Page 54: How To Read Lamp Energy

    Before you begin this procedure, your detector must be set up and configured, as described in Chapter During the start-up verification sequence, the 2998 PDA detector performs an erbium calibration. You can also start this procedure manually. Notes: •...

  • Page 55: Displaying The Rear-Panel Interface Connections

    A red LED symbol indicates no signal cable is connected to the terminal. To display 2998 PDA detector rear panel interface connections: In the console, select 2998 PDA Detector from the system tree. In the PDA detector information window, click Troubleshoot > Rear panel.

  • Page 56: Changing The Rear-Panel Interface Connections

    To change 2998 PDA detector rear panel interface connections: In the console, select 2998 PDA Detector from the system tree. In the PDA detector information window, click Troubleshoot > Rear Panel.

  • Page 57: Clearing Bubbles From The Flow Cell

    Flashing red Indicates that an error stopped the detector. The console's log records information about errors that cause operational failure 2998 PDA Detector Online Help See also: Constant red Indicates a detector failure that prevents further operation. Cycle power to the detector. If the LED is still steady red, contact your Waters service representative.

  • Page 58: Detector Troubleshooting

    4.2.4 Detector troubleshooting Table 4–3: 2998 PDA detector troubleshooting Symptom Possible cause Corrective action Both LEDs unlit No electrical power 1. Inspect line cord connections. 2. Test electrical outlet for power. Open (spent) or defective fuse Replace fuse (see page 51).
  • Page 59 Table 4–3: 2998 PDA detector troubleshooting (continued) Symptom Possible cause Corrective action Lamp light flashes red and Failed startup diagnostic tests Reseat and check alignment power light constant green of flow cell (see page 49). Flush the flow cell (see page 47).
  • Page 60 December 5, 2017, 715004753 Rev. B Page 60...

  • Page 61: Spectral Contrast Theory

    Spectral Contrast Theory The spectral contrast algorithm compares the UV/Vis absorbance spectra of samples that the detector collects. This chapter describes the theory on which the algorithm is based by 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.

  • Page 62: Representing Spectra As Vectors

    Representing spectra as vectors The spectral contrast algorithm uses vectors to quantify differences in the shapes of spectra, converting baseline-corrected spectra to vectors and then comparing the vectors. Spectral vectors have two properties: • Length – Proportional to analyte concentration. •...

  • Page 63: Vectors Derived From Multiple Wavelengths

    Finally, note that the length of the vectors is proportional to concentration. 5.2.2 Vectors derived from multiple wavelengths When absorbance ratios are limited to two wavelengths, the chance that two different spectra share an absorbance ratio is greater than if comparison is made using absorbance ratios at many wavelengths.
  • Page 64 Figure 5–3: Spectra that produce a large spectral contrast angle Spectral contrast angle: 62.3× Compound A Compound B Wavelength (nm) 5.3.1.1 Spectra with similar shapes In the following figure, the absorbance spectra of two compounds, A and B, are similar. They therefore produce a small spectral contrast angle (3.0).

  • Page 65: Differences Between Spectra Of The Same Compound

    5.3.2 Differences between spectra of the same compound Small but significant differences between absorbance spectra can result from factors other than the absorbance properties of different compounds. For example, multiple spectra of the same compound may exhibit slight differences because of detector noise, photometric error, high sample concentration, or variations in solvent conditions.

  • Page 66: Detector Noise

    5.4.1 Detector noise Statistical and thermal variations add electronic noise to the detector’s absorbance measurements. The noise, which manifests itself as fluctuations in the baseline, is known as baseline noise. The magnitude of any absorbance differences caused by statistical and thermal variations can be predicted from the instrument noise in the baseline region of a chromatogram.

  • Page 67: Threshold Angle

    5.4.4 Threshold angle In addition to computing spectral contrast angles, the spectral contrast algorithm also computes a threshold angle. The threshold angle is the maximum spectral contrast angle between spectra that can be attributed to nonideal phenomena. Comparison of a spectral contrast angle to its threshold angle can help determine whether the shape difference between spectra is significant.
  • Page 68 December 5, 2017, 715004753 Rev. B Page 68...

  • Page 69: A Safety Advisories

    Heed all warnings when you install, repair, or operate any Waters instrument or device. Waters accepts no liability in cases of injury or property damage resulting from the failure of individuals to comply with any safety precaution when installing, repairing, or operating any of its instruments or devices.

  • Page 70: Specific Warnings

    A.1.1.2 Biohazard warning The following warning applies to Waters instruments and devices that can process material containing biohazards, which are substances that contain biological agents capable of producing harmful effects in humans. To avoid infection with potentially infectious, human-sourced products,...

  • Page 71: Notices

    A.1.1.3 Biohazard and chemical hazard warning These warnings apply to Waters instruments and devices that can process biohazards, corrosive materials, or toxic materials. To avoid personal contamination with biohazards, toxic materials, or corrosive Warning: materials, you must understand the hazards associated with their handling.

  • Page 72: Warnings That Apply To All Waters Instruments And Devices

    Wear clean, chemical-resistant, powder-free gloves when handling samples. Requirement: Warnings that apply to all Waters instruments and devices When operating this device, follow standard quality-control procedures and the equipment guidelines in this section. Changes or modifications to this unit not expressly approved by the party Attention: responsible for compliance could void the user’s authority to operate the equipment.
  • Page 73 Manipulez les tubes en polymère sous pression avec precaution: Attention: • Portez systématiquement des lunettes de protection lorsque vous vous trouvez à proximité de tubes en polymère pressurisés. • Eteignez toute flamme se trouvant à proximité de l’instrument. • Evitez d'utiliser des tubes sévèrement déformés ou endommagés. •...
  • Page 74 警告：当有压力的情况下使用管线时，小心注意以下几点： • 当接近有压力的聚合物管线时一定要戴防护眼镜。 • 熄灭附近所有的火焰。 • 不要使用已经被压瘪或严重弯曲的管线。 • 不要在非金属管线中使用四氢呋喃或浓硝酸或浓硫酸。 要了解使用二氯甲烷及二甲基亚枫会导致非金属管线膨胀，大大降低管线的耐压能力。 경고: 가압 폴리머 튜브로 작업할 경우에는 주의하십시오. • 가압 폴리머 튜브 근처에서는 항상 보호 안경을 착용하십시오. • 근처의 화기를 모두 끄십시오. • 심하게 변형되거나 꼬인 튜브는 사용하지 마십시오. • 비금속(Nonmetallic) 튜브를 테트라히드로푸란(Tetrahydrofuran: THF) 또는 농축...

  • Page 75: Warnings That Address The Replacing Of Fuses

    警告： ユ ー ザ ー は、 製造元 に よ り 指定 さ れ て い な い⽅法で機器 を 使⽤す る と 、 機器が提供 し て い る 保証が無効 に な る 可能性が あ る こ と に 注意 し て 下 さ い。 Warnings that address the replacing of fuses The following warnings pertain to instruments and devices equipped with user-replaceable fuses.

  • Page 76: Electrical And Handling Symbols

    pour éviter tout risque d'incendie, remplacez toujours les fusibles par d'autres du Attention: type et de la puissance indiqués dans la rubrique "Remplacement des fusibles" du chapitre traitant des procédures de maintenance. Zum Schutz gegen Feuer die Sicherungen nur mit Sicherungen ersetzen, deren Vorsicht: Typ und Nennwert im Abschnitt "Sicherungen ersetzen"...

  • Page 77: Handling Symbols

    Symbol Description Safety ground Frame, or chassis, terminal Fuse Functional ground Input Output A.7.2 Handling symbols The following handling symbols and their associated statements can appear on labels affixed to the packaging in which instruments, devices, and component parts are shipped. Symbol Description Keep upright!
  • Page 78 Symbol Description Upper limit of temperature Lower limit of temperature Temperature limitation December 5, 2017, 715004753 Rev. B Page 78...

  • Page 79: B Specifications

    Allow the detector to warm for a minimum of two hours, and reach an equilibrium state Note: (stable baseline), before conducting the performance test. Physical specifications The following table lists the physical specifications for the 2998 PDA detector. Table B–1: Physical specifications Attribute...

  • Page 80: Performance Specifications

    Moisture Protection – Normal (IPXO) – IPXO means that no Ingress Protection against any type of dripping or sprayed water exists. The X is a placeholder that identifies protection against dust, if applicable. Performance specifications The following table lists the performance specifications for the 2998 PDA detector. Table B–4: Performance specifications...

  • Page 81: Optical Component Specifications

    Wet testing should be done with 90:10 water/acetonitrile, to minimize the effects of oxygen at 230 nm. 90:10 water/methanol can be substituted with appropriate solvent conditioning. Optical component specifications The following table lists the optical component specifications for the 2998 PDA detector. Table B–5: Optical component specifications...

  • Page 82: Flow Cell Specifications

    US patent number 5,883,721 Flow cell specifications The following table lists the flow cell specifications for the 2998 PDA detector. In the ACQUITY Arc system, the low-dispersion analytical flow cell is standard. In the ACQUITY Arc Bio system, the ®...

  • Page 83: C Solvent Considerations

    Solvent Considerations This appendix contains certain solvent considerations you must take into account when operating or maintaining the 2998 PDA Detector. To avoid chemical hazards, always observe Good Laboratory Practices Warning: when operating your system. Introduction C.1.1 Clean solvents Clean solvents provide reproducible results and permit you to operate with minimal instrument maintenance.

  • Page 84: Use Buffers

    C.1.5 Use buffers When you use buffers, dissolve salts first, adjust the pH, then filter to remove insoluble material. C.1.6 Tetrahydrofuran When using unstabilized tetrahydrofuran, ensure that your solvent is fresh. Previously opened bottles of tetrahydrofuran contain peroxide contaminants, which cause baseline drift. Tetrahydrofuran contaminants (peroxides) are potentially explosive if Warning: concentrated or taken to dryness.

  • Page 85: How To Use Miscibility Numbers

    Table C–1: Solvent miscibility (continued) Boiling  Cutoff Polarity Viscosity Miscibility Point C Solvent CP, 20 C Index Number (M) (nm) (1 atm) Butyl alcohol 3.00 117.7 ––- –– Butanol 3.01 177.7 –– Tetrahydrofuran 0.55 66.0 Ethyl acetate 0.47 77.1 1-propanol 2.30 97.2...

  • Page 86: Buffered Solvents

    Some solvents prove immiscible with solvents at both ends of the lipophilicity scale. These solvents receive a dual M-number: • The first number, always lower than 16, indicates the degree of miscibility with highly lipophilic solvents. • The second number applies to the opposite end of the scale. A large difference between these two numbers indicates a limited range of miscibility.

  • Page 87: Solvent Viscosity

    Figure C–1: Minimum bend radius for stainless steel tubing (continued) Tubing size (OD) Minimum bend radius 1/8-inch tubing 1/2-inch Solvent viscosity Generally, viscosity is not important when you are operating with a single solvent or under low pressure. However, when you are running a gradient, the viscosity changes that occur as the solvents are mixed in different proportions can result in pressure changes during the run.

  • Page 88: Solvent Degassing Methods

    C.7.1.1 Effects of intermolecular forces Nonpolar gases (N , CO , He) are more soluble in nonpolar solvents than in polar solvents. Generally, a gas is most soluble in a solvent with intermolecular attractive forces similar to those in the gas (like dissolves like). C.7.1.2 Effects of temperature Temperature affects the solubility of gases.

  • Page 89: Solvent Degassing Considerations

    C.8.3 Solvent degassing considerations Select the most efficient degassing operation for your application. To remove dissolved gas quickly, consider the following. C.8.3.1 Sparging Helium sparging gives stable baselines and better sensitivity than sonication in a detector, and prevents reabsorption of atmospheric gases. Use this method to retard oxidation when you are using tetrahydrofuran or other peroxide-forming solvents.

  • Page 90: Mixed Mobile Phases

    Reagent A, 1 vial/liter MES, 10 mM, pH 6.0 Waters PIC Reagent B-6, 1 vial/liter Potassium phosphate, Waters PIC Reagent B-6, low UV, monobasic, 10 mM 1 vial/liter dibasic, 10 mM Sodium acetate, 10 mM Waters PIC Reagent D-4, 1 vial/liter December 5, 2017, 715004753 Rev.
  • Page 91 C.9.3 Wavelength selection for chromophore detection Certain functional groups found in most compounds absorb light selectively. These groups, known as chromophores, and their behavior can be used to categorize the detection of sample molecules. The table below lists some common chromophores, and their detection wavelengths ( ...
  • Page 92 Table C–4: Electronic absorption bands of representative chromophores (continued)*    Chemical  Chromophore (nm) Configuration (L/m/cm) (nm) (L/m/cm) Allene —(C=C) 4 — 52,000 Allene —(C=C) 5 — 118,000 Allene —(C=C) 2 — 230-260 3000-8000 (alicyclic) Ethylenic/ C=C—CC 6,500 Acetylenic Ethylenic/ C=C—C=N...
  • Page 93 Table C–5: Mobile phase absorbance measured against air or water (continued) Absorbance at specified wavelength (nm) 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 >2.5 >2.5 >2.5 >2.5 >2.5 >2.5 >2.5 >2.5 1.45 tetrahydrofuran (THF), old Acids and bases...
  • Page 94 7.0 Tris HCl, 20 mM, 1.80 1.90 1.11 0.43 0.13 <0.01 — — — — pH 8.0 ® ® Waters reagents PIC A, 1 vial/L 0.67 0.29 0.13 0.05 0.03 0.02 0.02 0.02 0.02 <0.01 PIC B6, 1 vial/L 2.46 2.50...

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