HPLC Troubleshooting Guide
安捷伦液相维护
25
Agilent 四元／二元/单元泵日常维护
多通道梯度阀（MCGV）操作要点
当盐溶液和有机溶剂混合时，盐溶液可能与有机溶剂 完全混溶，而不会出现 沉淀。但是在梯度阀的混合点，但是 在梯度阀的混合点，重力作用使盐颗粒 沉淀下来。通常，阀 的A通道用于水相/盐溶液，而泵的B通道用于有机溶 剂。如果 使用的是这样的配置，盐将落回到盐溶液中，并被溶解。当泵 使用 的不同配置（例如，D-盐溶液，A-有机溶剂）时，盐可 能会落到有机溶剂 中，从而出现问题。 Agilent 1100四元泵中使用盐溶液和有机溶剂时，建 议将盐溶液接到下面的 口上，有机溶剂接到上面的梯度阀口 上。有机溶剂通道最好直接在盐溶液通 道的上面。建议定期 用水冲洗所有MCGV通道，以去除在阀口上可能出现的盐 沉淀 。
用二次蒸馏水彻底冲洗过滤器。建议不使用超声波清洗机清洗。
将过滤器重新装好。 建议定期清洗溶剂过滤器及溶剂瓶，每三个月至少清洗一次。
注意：不要使用没有安装溶剂过滤器的系统．可能会严重堵塞系统
11
Agilent 1100 在线脱气机日常维护
Question?
1.如何简单判断脱气机脱气是否正常？ 2.如何简单判断脱气机腔体或溶剂过滤头是否 堵塞？
12
Agilent 1100 在线脱气机日常维护
Question?
1.如何简单判断脱气机脱气是否正常？ 打开Purge阀，设定泵的流速2ml/min，提 起当前所使用溶剂瓶内的溶剂过滤头，使之脱 离液面一小段时间，此时溶剂传送管内会产生 一小段气泡，放下过滤头，让此段气泡通过脱 气机，如果脱气机正常，气泡应消失或缩小。 2.如何简单判断脱气机腔体或溶剂过滤头是否 堵塞？ 关闭泵，拧开脱气机出口或比例阀入口管线, 此时溶剂会因为重力流出，脱气机或溶剂过滤 头堵塞时，溶剂会流出不畅或不流出。
戴安-hilic柱说明书
forAcclaim®Mixed-Mode HILIC-1ColumnProduct ManualforACCLAIM® Mixed-Mode HILIC-1 Column5µm, 4.6 x 250mm, P/N 0668445µm, 4.6 x 150mm, P/N 0668435µm, 2.1 x 150mm, P/N 0668473µm, 3.0 x 150mm, P/N 0700903µm, 3.0 x 50mm, P/N 0719123µm, 2.1 x 150mm, P/N 070091 ACCLAIM® Mixed-Mode HILIC-1 Guard4.3 x 10mm (set of 2), P/N 0668452.1 x 10mm (set of 2), P/N 0668463.0 x 10mm (set of 2), P/N 0719134.6 x 10mm (set of 2), P/N 069706© 2009 Dionex CorporationDocument No. 065198Revision 02July 2009TABLE OF CONTENTSSECTION 1 – Introduction (3)1.1. Specifications and Recommended Operating Conditions (3)1.2. Physical (4)1.3. Acclaim Mixed-Mode HILIC-1 Products (4)SECTION 2 – INSTALLATION: Step-by-Step User Guide (5)SECTION 3 – Considerations in Method Development (7)3.1. HILIC Mode vs. Reversed-Phase Mode (7)3.2. Selection of Organic Solvents (7)3.3. Buffer Types (7)3.4. Mobile Phase pH (7)3.5. Isocratic or Gradient Method (7)SECTION 4 – Column Care (8)4.1. Column storage (8)4.2. Operating pH range: pH 2.5 to 7.5 (8)4.3. Operating temperature limit: 50 ºC (8)4.4. Pressure limit < 4000 psi (8)4.5. Flow rate (8)4.6. Column washing procedure (8)SECTION 5 – Frequent Asked Questions (9)SECTION 6 – Applications (10)6.1. Dual Operation Modes: Reversed-phase and HILIC (10)6.2. Analysis of Urea (11)6.3. Complementary Selectivity: Reversed-phase and HILIC (12)6.4. Dependency of Retention on Mobile Phase Organic Content:Comparison between the Acclaim HILIC and LiChrosorb Diol (13)6.5. Analysis of NEODOL 25-12 (14)6.6. Analysis of Alkylphenol Ethoxylate (15)6.7. Separation of Polyethylene Glycols (16)SECTION 7 – Troubleshooting Guide (17)7.1. High Backpressure (17)7.1.1. Finding the Source of High System Pressure (17)7.1.2. Contaminated guard or analytical column (17)7.2. High Background or noise (17)7.2.1. Preparation of Mobile Phases (17)7.3. Poor Peak Resolution (17)7.3.1. Loss of column Efficiency (17)SECTION 1 – INTRODUCTIONThe Acclaim Mixed-Mode HILIC-1 column is a silica-based packing material that incorporates both Reversed-Phase (RP) and Hydrophilic Interaction Liquid Chromatography (HILIC) properties. Unlike either traditional RP or HILIC stationary phases, the packing features an alkyl long chain with a hydrophilic polar terminus, and demonstrates great potentials for separating a wide range of both highly polar and non-polar molecules, in either RP mode or HILIC mode.The Acclaim Mixed-Mode HILIC-1 columns consist of 5-µm or 3-µm high-purity, porous, spherical silica particles with 120Å diameter pores bonded with proprietary functional groups, which provide unsurpassed resolution and peak symmetry for a variety of polar and non-polar molecules.The main features of the Acclaim HILIC-1 column include1. Operates in both reversed-phase and normal phase modes.2. Retains highly polar molecules that would be un-retained by reversed-phase chromatography.3. Unique selectivity, complementary to reversed-phase columns.4. Higher hydrophobic retention compared to the conventional Diol columns.5. Broader application range than conventional Diol columns.1.1.Specifications and Recommended Operating ConditionsShipping Solution 60 / 40 Acetonitrile / 100mM Ammonium AcetateStorage Solution: 60 / 40 Acetonitrile / 100mM Ammonium Acetateor HILIC mobile phaseBuffer pH Range: pH 2.5 - 7.5Temperature Range < 50°CStationary Phase ParticlesizeColumnDimensionsP/NMax RecommendedPressureTypical Flow Rate2.1x150 mm0700915800 psi0.2 - 0.5 mL/min3.0x50 mm0719124500 psi0.4 - 1.0 mL/min 3 µm3.0x150 mm0700905800 psi0.4 - 1.0 mL/min2.1x150 mm0668475800 psi0.2 - 0.5 mL/min4.6x150 mm0668435800 psi0.8- 2.0 mL/minMixed-Mode HILIC-15 µm4.6x250 mm0668445800 psi0.8 - 2.0 mL/min1.2.PhysicalBonding Chemistry: Proprietary alkyl diol Silica Substrate: Spherical, high-purity Particle size 5 µm and 3µmSurface area 300 m2/gPore size 120 Å1.3.Acclaim Mixed-Mode HILIC-1 ProductsAcclaim Mixed-Mode HILIC-1 ParticlesizeColumnDimensionsP/N2.1x150 mm 0700913.0x50 mm 0719123 µm3.0x150 mm 0700902.1x150 mm 0668474.6x150 mm 066843Analytical5 µm4.6x250 mm 0668444.3 x 10mm 066845 Requires Holder p/n 0594562.1 x 10mm 066846 Requires Holder V-2 p/n 0695803.0 x 10mm 071913 Requires Holder V-2 p/n 069580 Guard 5µm4.6 x 10mm 069706 Requires Holder V-2 p/n 069580SECTION 2 – INSTALLATION: STEP-BY-STEP USER GUIDEStep 1 - Validating column performanceDionex recommends that you perform an efficiency test on your Acclaim Mixed-Mode HILIC column before use. The purpose of column performance validation is to ensure no damage has occurred during shipping. Test the column using the conditions described on the Quality Assurance (QA) Report enclosed in the column box. Repeat the test periodically to track the column performance over time. Note that slight variations may be obtained on tow different HPLC systems due to system electronic, plumbing, operating environment, reagent quality, column conditioning, and operator technique.Step 2 - Mobile phase preparationObtaining reliable, consistent and accurate results require mobile phases that are free of ionic and spectrophotometric impurities. Chemicals, solvents and de-ionized water used to prepare mobile phase should be of the highest purity available. Maintaining low trace impurities and low particle levels in mobile phases helps to protect your columns and system components. DIONEX cannot guarantee proper column performance when the quality of the chemicals, solvents and water used to prepare the mobile phase has been compromised.De-ionized WaterThe de-ionized water used to prepare the mobile phase should be Type 1 Reagent Grade Water, or HPLC Grade Water. The de-ionized water should be free of ionized impurities, organics, microorganisms and particulate matter larger than 0.2 µm. Many commercial water purifiers are designed for HPLC applications and are suitable for these applications.NOTE Degas the aqueous component of the mobile phase and then add the solvent component. Avoid excessive purging or degassing of mobile phases containing solvents, if possible, since the volatile solvent can be 'boiled' off from the solution.SolventsThe solvents used must be free from ionic and UV-absorbing impurities. Use of ultrahigh purity solvents, HPLC grade, will usually ensure that your chromatography is not affected by impurities in the solvent.Depending on specific application, the mobile phase system consists of an organic modifier (e.g. acetonitrile or methanol) and an aqueous portion (e.g. D.I. water, or ammonium acetate or phosphate buffer). Both pre-mixed and proportioning valve generated mobile phases give satisfactory results. The use of proportioning valve provides better flexibility in method optimization, while the pre-mixed mobile phase provides more reproducible results.Mobile Phase for Column Performance Test:Pre-mixed mobile phase: mix 400 g of D.I. water and 468 g of acetonitrile.Proportioning valve generated: set up the gradient pump as 40/60 v/v D.I. water/ acetonitrile.NOTEThese two mobile phases could give slightly different results due to the ways they are prepared.Step 3 - Set up the LC systemUse a standard LC system equipped with a LC pump, a column oven, a UV detector, and an injector (or an autosampler). The system should be thoroughly primed before use.Step 4 - Condition the columnEach new column is shipped in the mobile phase used for the column performance test. Depending on different separation modes, the column should be conditioned differently.For HILIC mode (>70% acetonitrile in the mobile phase) applications, the column should be purged with 50% acetonitrile in D.I. water (v/v) for approximately 10 column volumes, followed by equilibration with the desirable mobile phase thoroughly (~20 to 50 column volumes depending on the aqueous content in the mobile phase) before any injection is made.For RP mode (<70% acetonitrile in the mobile phase) applications, the column should be purged with 50% acetonitrile in D.I. water (v/v) for approximately 10 column volumes, followed by equilibration with the desirable mobile phase thoroughly (~20 column volumes) before any injection is made.When switching to a different mobile phase, make sure that the new mobile phase is compatible with the existing mobile phase in the column to avoid column clogging due to precipitation. A good practice is to purge the column with 50% acetonitrile in D.I. water (v/v) for approximately 10 column volumes before switching to the new mobile phase.Step 5 - Reproduce the chromatogram in the Lot Validation ReportPerform the column QA test using the conditions described in the QAR (Appendix), and compare the result with the reported values. The column should be fully equilibrated before any injection. At least three injections should be made until reproducible result is obtained.NOTE Due to various reasons, such as difference of LC systems, mobile phases, oven temperature control, etc, you may observe somewhat different separation from that in the report.Once you are satisfied with the column performance report result, the column is ready for real applications.SECTION 3 – CONSIDERATIONS IN METHOD DEVELOPMENT3.1.HILIC Mode vs. Reversed-Phase ModeThe selection of separation mode depends on the type of the analyte molecules and requirement of the specific application. In general, if a highly polar molecule is the analyte of interest, HILIC mode (high organic in the mobile phase) should be considered. In addition, HILIC mode can be used to determine the degree of ethoxylation (EO) of a nonionic surfactant. For other applications, RP separation may be considered. However, it is recommended that a conventional RP column (C18, C8, or polar-embedded phase) be tried first for regular RP applications. The Acclaim HILIC column has lower hydrophobicity than C8 columns but much higher hydrophobic retention than conventional Diol columns because of its unique surface chemistry, so that selectivity different from RP columns is possible.3.2.Selection of Organic SolventsFor HILIC applications, acetonitrile is preferred as the organic modifier.For RP applications, both acetonitrile and methanol can be used depending on the application.In addition, this column is compatible with traditional normal-phase solvent, such as ethanol, iso-propanol, ethyl acetate, hexane, heptane, etc.3.3.Buffer TypesThe selection of buffer depends on the detection method and pH requirement.Ammonium acetate (or formate) buffer is the preferred buffer system because of its applicability to both RP and HILIC separation modes, compatibility with UV (> 230 nm), ELS detector and MS, high solubility in organic solvent, familiarity to most chromatographers.Volatile organic acids, such as acetic acid, formic acid, and TFA, can also be used to control the mobile phase pH, and share the similar benefits of ammonium acetate (formate) buffer.Phosphate buffers are ideal for applications that require low UV background, and it usually provides somewhat better peak shapes for charged molecules, compared to acetate buffer. Although there is no problem in RP application(organic content < 70%), phosphate buffers tend to precipitate in high organic condition.3.4.Mobile Phase pHMobile phase pH needs to be controlled to adjust selectivity and obtain reproducible results of charged molecules.3.5.Isocratic or Gradient MethodIsocratic methods are suitable for simple and/or well-defined application. When dealing with unknown samples, or a sample consisting of molecules with dramatically different hydrophilicity or hydrophobicity, a gradient method is often advantageous.SECTION 4 – COLUMN CARE4.1. Column storageThe column can be stored in the mobile phase for short period of time (e.g. overnight). If not in use for longer than one week, it is recommended to store the column in a solution with higher organic content, such as 70/30 v/v acetonitrile (or methanol)/20-100 mM ammonium acetate or D.I. water at a pH between 3.5 and 5.5.4.2. Operating pH range: pH 2.5 to 7.5The column lifetime depends heavily on the chromatographic condition. To obtain better column lifetime, it is highlyrecommended to use "silica friendlily" mobile phases, such as a buffer at a pH between pH 3 to 7. Although compatible with 100% aqueous mobile phase, it is not recommended to use this column extensively in such condition. The mobile phase pH should be controlled between 3.5 and 6 if more than 80% buffer is present in the mobile phase. If used in HILIC conditions, the column can be used in pH 2 to 8 range.4.3. Operating temperature limit: 50 ºCAlthough our experimental results indicate that the column can be used at 50 °C, a lower operating temperature (20 - 40 °C) is recommended for routine separations.4.4. Pressure limitIt is extremely important not to impose sudden column pressure surge. The maximum recommended pressures are listed in section 1.1, the use of lower operating pressures will extend column lifetime.4.5. Flow rateThe typical operating flow rates are listed in section 1.1, higher flow rate can be used for faster analysis as long as the pressure limit is not exceeded.4.6. Column washing procedureAll samples should be pre-treated and filtered before being injected on the column. In the event of column washing practice is needed, the following procedure can be used as a guideline: For a 4.6 mm i.d. column:1. Wash the column with D.I water /acetonitrile v/v 50/50 for 10 column volumes at a flow rate between 0.5 to 1mL/min.2. Wash the column with 0.1 M ammonium acetate pH 4 to 5 /acetonitrile v/v 50/50 for 20 column volumes at a flowrate between 0.5 to 1 mL/min.3. Wash the column with 0.1 M ammonium acetate pH 4 to 5 /acetonitrile v/v 10/90 for 20 column volumes at a flowrate between 0.5 to 1 mL/min.4. Wash the column with 0.1 M ammonium acetate pH 4 to 5 /acetonitrile v/v 50/50 for 20 column volumes at a flowrate between 0.5 to 1 mL/min.5. Before any injection is made, the column should be equilibrated with the mobile phase thoroughly.NOTEFor a 2.1 mm i.d. column, the flow rate should be reduced to 20% of that for 4.6 mm i.d column.NOTEIf above treatment fails to revive the column, the column should be replaced.SECTION 5 – FREQUENT ASKED QUESTIONS1. What is the difference between the Acclaim Mixed-Mode HILIC-1 column and conventional HILIC (Diol) column? The Acclaim HILIC-1 column is based on a new silica-based mixed-mode stationary phase that incorporates both RP and HILIC properties. Unlike either traditional RP or HILIC stationary phases, the new packing features an alkyl long chain with a hydrophilic polar terminus, and demonstrates broader applications for separating a wide range of both highly polar and non-polar molecules, in either RP mode or HILIC mode (Figure 1). By comparison, conventional Diol columns have significantly decreased hydrophobicity (Figure 2), and have much narrower application range.2. Why do I need an Acclaim Mixed-Mode HILIC-1 column?The Acclaim HILIC-1 column combines both RP and HILIC characteristics. It can be used in either HILIC conditions or RP conditions the same way as conventional Diol columns or RP columns, without special requirements. More importantly, the optimal balance between the hydrophilic and hydrophobic portions on the column surface leads to unique selectivity that neither conventional Diol columns nor RP columns can provide.3. When do I need an Acclaim Mixed-Mode HILIC-1 column?You should consider using an Acclaim HILIC-1 column when1) Regular RP (e.g. C18, C8, polar-embedded phase, etc) and HILIC (e.g. Diol, CN, amino, silica) columns fail toprovideresults;satisfactory2) You are dealing with highly polar molecules, or very hydrophobic molecules;3) You need orthogonal selectivity to complement the primary method;4) You are analyzing ethoxylated surfactants (Figures 6 - 8).4. What factors should I consider for method development using this column?During method development, the following factors should be considered:1) Separation mode2) Type of organic modifier3) Aqueous content in the mobile phase4) Mobile phase pH control5) Temperature5. What mobile phases should I use with this column?The new column is compatible with any mobile phases for HILIC or Normal-Phase separations provided that the pH requirement is met.The new column is compatible with any mobile phases for Revered-Phase separations provided that the pH requirement is met. However, for typical RP applications, a RP column (C18 or C8) should be tried first.Please refer to "Section 3 Considerations in Method Development" for more details.6. What should I do before starting using Acclaim Mixed-Mode HILIC-1 column?Read this User Guide carefully, and contact Dionex Technical Support if you have any questions regarding using this column.7. Can I use this column in normal-phase mode?Yes. The column is compatible with traditional normal-phase solvent, such as ethanol, iso-propanol, ethyl acetate, hexane, heptane, etc.8. How to store the column?The column can be stored in the mobile phase for short period of time (e.g. overnight). If not in use for longer than one week, it is recommended to store the column in a solution with higher organic content, such as 70/30 v/v acetonitrile (or methanol)/20-100 mM ammonium acetate or D.I. water at a pH between 3.5 and 5.5.SECTION 6 – APPLICATIONS6.1.Dual Operation Modes: Reversed-phase and HILICAUMinutesColumn: Acclaim Mixed-Mode HILIC-1, 5 µmDimensions: 4.6 x 150 mmMobile Phase: CH3CN/0.1 NH4OAc, pH5.2v/v 52/48 for RP mode;v/v 92/8 for HILIC mode°CTemperature: 30Flow Rate: 1 mL/minInj. Volume: 10 µLDetection: UV @ 254 nmeach)ppmPeaks: (100Cytosine1.Naphthalene2.6.2.Analysis of Urea486210MinutesmV95/5 v/v CH 3CN/0.1 M NH 4OAc, pH4.597/3 v/v CH 3CN/0.1 M NH 4OAc, pH4.597.5/2.5 v/v CH 3CN/0.1 M NH 4OAc, pH4.5Column: Acclaim Mixed-Mode HILIC-1, 5 µm Dimensions: 4.6 x 150 mm Mobile Phase: See chromatograms for details Temperature: 30°C Flow Rate: 1 mL/min Inj. Volume: 10 µL Detection: Sedex 85 ELS detector (gain - 9, 50ºC) Sample: Urea (0.1% in 90% CH 3CN)6.3.Complementary Selectivity: Reversed-phase and HILIC81612420MinutesAUCH 3CN/0.1NH 4OAc,pH5.2 v/v 97.5/2.5CH 3CN/0.1NH 4OAc,pH5.2 v/v 60/40123213Acclaim 120C8Acclaim Mixed-Mode HILIC-1Column: 5 µm Dimensions: 4.6 x 150 mm Mobile Phase: CH 3CN/0.1 NH 4OAc, pH5.2 Temperature: 30 °C Flow Rate: 1 mL/min Inj. Volume: 5 µL Detection: UV @ 254 nm Peaks:1. Toluene2. Caffeine3. Benzoic acid6.4.Dependency of Retention on Mobile Phase Organic Content:Comparison between the Acclaim HILIC and LiChrosorb DiolColumn: Acclaim Mixed-Mode HILIC-1, LiChrosorb Diol, 5 μmMobile Phase: CH3CN/0.1 NH4OAc, pH5.2Flow Rate: 1 mL/minDetection: UV @ 254 nmDimensions: 4.6 x 150 mmTemperature: 30°CInj. Volume: 5 µL6.5.Analysis of NEODOL 25-1281612420MinutesC12C13C14C15C12C13C14C15Acclaim HILICAcclaim 120C8NEODOL 25–12R (OCH 2CH 2)n OH R = C 12 to C 15n ~Column: 5 µm Dimensions: 4.6 x 150 mm Mobile Phase: A - CH 3CN, B - D.I. H 2O Gradient: 30% to 80% A in 15 min,then hold for 5 min Temperature: 30 °C Flow Rate: 1.5 mL/min Inj. Volume: 10 µLDetection: ELS Detector Sample: NEODOL 25-12 (0.2%)6.6.Analysis of Alkylphenol Ethoxylate122418630MinutesAUIGEPAL CA-630n ~9C 8H 17(OCH 2CH 2)n OHAcclaim HILICLiChro sorb DiolAcclaim 120 C8Column: See chromatograms for details, 5 µm Dimensions: 4.6 x 150 mm Mobile Phase: 99/1 v/v CH 3CN/0.1 M NH 4OAc, pH5.2 Temperature: 30 °C Flow Rate: 1.0 mL/min Inj. Volume: 10 µL Detection: UV @ 225 nm Sample: IGEPAL CA-630 (0.1%)6.7.Separation of Polyethylene Glycols81612420MinutesmVPEG-400PEG-1000PEG-2000PEG-3400PEG-6000Column: Acclaim HILIC, 5 µm Dimensions: 4.6 x 150 mm Mobile Phase: A - MeOH, B - D.I. H 2O Gradient: 20% to 95% A in 20 min Temperature: 30 °C Flow Rate: 1 mL/min Inj. Volume: 25 µLDetection: ELSD Samples: Various PEGs (0.04% each)SECTION 7 – TROUBLESHOOTING GUIDEThe following instruction should help you to locate and eliminate problems traceable to hardware and chemistry issues. Please keep in mind that some problems may be due to other reasons, such as sample contamination, poor water quality, etc. If you cannot solve your problems with the help of this manual, please contact you local Dionex customer support specialist.7.1.High Backpressure7.1.1.Finding the Source of High System PressureIf the system pressure is excessively high, determine the cause of the high pressure. The system should be used with a high-pressure in-line filter for mobile phases. The filter should be positioned between the gradient pump pressure transducer and the injection valve. Make sure you have a high-pressure in-line filter in place and that it is not contaminated.A.Make sure that the pump is set to the correct flow rate. Higher than recommended mobile phase flow rates will causehigher pressure. Measure the pump flow rate if necessary with a stop watch and graduated cylinder.B.Determine which part of the system is causing the high pressure. It could be a piece of tubing that has plugged,collapsed tubing walls from over tightening, an injection valve with a plugged port, a column with particulatesplugging the bed support, a plugged High-pressure In-line filter, or detector cell. To identify which part of thechromatographic system is causing the problem, disconnect the pump fluid line from the injection valve and turn the pump on. Watch the pressure; it should not exceed 50psi (0.34MPa). Continue adding the system components back into the fluid path, one by one, while watching the system pressure.7.1.2.Contaminated guard or analytical columnIf the column is the cause of the back pressure, replace the guard column.7.2.High Background or noise7.2.1.Preparation of Mobile PhasesA.Ensure the mobile phase is made correctly.B.Ensure the eluents are made from chemicals with the recommended purity.7.3.Poor Peak ResolutionPoor peak resolution can be due to any or all of the following factors.7.3.1.Loss of column EfficiencyA.Extra-column system effects can result in sample band dispersion, decreasing peak efficiencies. Make sure you areusing tubing with an i.d. of no greater than 0.010" (0.007" for 2.1mm) to make all liquid line connections between the injection valve and the detector cell inlet on standard bore (4-mm) systems. Check for leaks.B.Guard column / column needs to be replaced.。
液相常用语中英文对照
Table of Contents（目录）How to Use This Guide （如何使用本指南）Chapter 1 Introduction to System Troubleshooting（系统故障检修介绍）1.1 Get the Facts（获取真相）1.2 Check Simple Things First（从简单的方面检查开始）1.3 Compare System Performance to Established Benchmarks（从比较系统性能到建立基准）1.4 Identify Possible Causes（确定可能原因）1.5 Use a Systematic Troubleshooting Approach（使用一种系统内部的故障检修方法）1.6 Getting Help（获取帮助）1.7 Troubleshooting References（故障检修参考书目）Chapter 2 Troubleshooting an LC System （检修一个液相色谱系统）2.1 Initial Survey of System Problems（系统问题的最初调查）2.2 Isolating System Problems（分离系统问题）2.2.1 System Pressure（系统压力）2.2.2 Baseline Noise（基线噪音）2.2.3 Changes in Chromatographic Resolution or Results（色谱分离度或结果的改变）Chapter 3 Troubleshooting System Components（检修系统构件）3.1 Pump Troubleshooting（色谱泵检修）3.1.1 Pump Troubleshooting T able（色谱泵检修表格）3.1.2 Isolating a Defective Pump Head (Modified Ramp Test)（分离一个受损泵头）3.2 Manual Injector Troubleshooting （进样器检修手册）3.3 Autoinjector Troubleshooting （自动进样器检修）3.4 Column Troubleshooting （色谱柱检修）3.5 Detector Troubleshooting（检测器检修）3.6 Data-Handling Device Troubleshooting（数据处理装置检修）Chapter 4 Good Chromatography/ Operating Practices（良好的色谱操作规范）4.1 Mobile Phase Preparation and Use（流动相制备和使用）4.1.1 Mobile Phase Preparation （流动相制备）4.1.2 Solvent Degassing（溶剂脱气）4.1.3 Solvent Use（溶剂使用）4.1.4 Solvent Changeover Practices（溶剂转换规范）4.2 System Plumbing （泵输送系统）4.2.1 Tubing Connection Practices （管道连接规范）4.2.2 Cutting Tubing（切割管路）4.3 Chromatographic Performance Tests（色谱性能测试）4.3.1 Measuring Resolution（测量分离度）4.3.2 Measuring Capacity Factor (k') （测量容量因子k'）4.3.3 Measuring Selectivity（测量选择性）4.3.4 Measuring Column Efficiency (N) （测量柱效（N））4.4 Measuring System Bandspreading（测量系统拖尾因子）Appendix A （附录A）Reference Information（参考信息）A.1 Solvent Properties（溶剂属性）A.1.1 Solvent Properties Table （溶剂属性表）A.1.2 Using Miscibility Numbers (M-Numbers) （使用可混和性编号（M-编号））A.2 Refractive Index of Common Solvents（常见溶剂的折射指标）.A.3 Solvent UV Cutoffs （溶剂紫外离群值截断点）A.4 Wavelength Selection for Chromophore Detection（色谱检测器的波长选择）A.5 Column Backpressure （柱后压）Waters公司液相色谱资料系列－ⅡHPLC Troubleshooting Guide （HPLC故障检修指南）本资料为英文版本，文件大小为836KB，PDF格式，全书共68页，编排为目录、主题内容和附录三部分，带书签和搜索功能，由美国Waters公司专家Uwe D. Neue编写，内容包括52个主题。
Agilent 1100 HPLC及消耗品简介
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四元泵
使用2，4，8mm内径的柱子 流量范围从 0.2 到 10 ml/min 应用于研究和常规分析
真空脱气机为标准配置。
Agilent Technologies
2003 LC 色谱柱
溶剂传输系统
往复活塞泵(单向阀)
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21页
含有过氧化物的色谱级醚必须用氧化铝干燥剂。尤其是使用折光 示差检测器时必须考虑
在有机溶剂中的有机酸溶液。例甲醇中1%乙酸会腐蚀不锈钢 含有强络合剂的溶液。
四氯化碳和异丙醇或THF混合液。
免费服务电话: 800-8203278
Agilent Technologies
2003 LC 色谱柱
Name Acetic Acid Acetone Acetonitrile Benzene Butyl Alcohol Carbon Tetrachloride Chloroform Cyclohexane Cyclopentane Dichloroethane Dichloromethane Dimethylformamide Dimethyl Sulfoxide Dioxan Ethylacetate Ethyl Alcohol Di-Ethylether Heptane Hexane Methyl Alcohol Methylethyl Ketone I-Octane Pentane I-Propyl Alcohol Di-Propylether Tetrachloroethane Tetrahydrofuran Toluene Trichloroethane Water Xylene
2003 LC 色谱柱
20页
戴安U3000-HPLC操作流程CM6.8
戴安Ultimate 3000系列HPLC标准操作规程一、准备工作：1、面板操作指南：1.1、如何进入菜单：使用仪器中配置的感应笔在仪器前面板显示屏下的白色定位点前约0.5cm处悬停约1秒或轻触，四个定位点的上方将显示相应的菜单功能，按需要进行相应的选择即可，常规参数的选择（或进入）使用“SELECT或OK”，返回上一层按“BACK”，“＜、∧、＜、∨”分别为光标移动键。
感应笔触任一定位点将直接显示出“常用功能菜单”，对于未显示的功能，则需要先选择“MENU”进入主菜单，再进一步进行选择。
1.2、各设备的主要功能：1.2.1、泵：A、泵开关：使用感应笔调出现常用功能菜单后，直接选择“FLOW ON”功能，即开启泵马达；在启动后，再次调出菜单后显示的“FLOW OFF”为关闭泵选项。
B、溶剂选择：使用感应笔调出常用功能菜单后，选择“MENU”，进入后再选择“CONTROL”功能，进入子菜单后，移动选项到相应的溶剂“A%、B%、C%、D%”进入后再调节相应的值即可。
在当前设置流动相比例不足100%，系统会自动与前一项进行分配，总和为100%。
C、流速的调节：使用感应笔调出常用功能菜单后，直接选择“SET FLOW”然后通过移动选项进行流速的设置。
注，数值不能自动进位，故设置时应确认显示的值为最终的值。
D、PURGE：使用感应笔调出常用功能菜单后，直接选择“PURGE”即执行，再次选择时即为关闭，当未做选择时系统会在执行5分钟后自动停止。
1.2.2、紫外检测器：A、灯开关：在使用感应笔调出常用功能菜单后，选择“MENU”进入子菜单后，再选择“GENERAL SETTING”，进入后，再进入下一级子菜单，通过感应笔移动选择“UV LAMP”，进入后再选择“TOGGLE”则打开氘灯；再选择“Vis LAMP”时，相应的操作打开钨灯。
B、波长选择：在调出常用功能菜单后，选择“WAVELENGHT”选择，再选择“WAVELENGHT 1”进入，通过感应笔移动光标选择修改检测波长。
HPLC保养
Female End Fitting 旋好尾端螺母 Male End Fitting 旋好尾端螺头
Split peaks 裂峰 Peak tailing 峰拖尾 Broad peaks 宽峰 Poor efficiency 低柱效
Many peak shape issues also combinations-I.e. Broad and tailing or tailing with increased retention许多峰形问题 是结合在一起的-例如: 展宽和拖尾或拖尾和保留时间增加
HPLC Column and System Troubleshooting
色谱柱和色谱系统的故障检修
What Every HPLC User Should Know
请访问中国色谱网()
Hale Waihona Puke 每个HPLC使用者应该知道什么
2014-7-10 1
HPLC Components HPLC 的组成
峰拖尾、变宽和柱效降低
Can be caused by: 可能的原因
Column “secondary interactions” 柱“次级效应” Column void 柱塌陷 Column contamination 柱污染 Column aging 柱老化 Column loading 柱负荷超载 Extra-column effects 柱外效应
Waters XBridge
非常感谢您选择Waters XBridge™色谱柱。
XBridge™填料设计精良，能在酸性和碱性流动相条件下获得良好的峰形、达到高柱效并展现出色的稳定性。
所有XBridge™填料均由通过cGMP和ISO 9001: 2000认证的工厂使用超纯试剂生产。
每批XBridge™填料都采用酸性、碱性和中性分析物进行了色谱测试，结果均符合严格的合格标准，可确保色谱柱具有出众的重现性能。
每根色谱柱都经过单独测试，并随附性能测试色谱图以及合格证书。
目录I. 入门指南a.色谱柱安装b.色谱柱平衡c.初始柱效测定II. 色谱柱使用a. 保护柱b.样品制备c. pH操作限值d.溶剂e.压力f.温度III. 放大/缩小等度方法IV. 故障排除V. 色谱柱清洗、再生和储存a.清洗与再生b.储存VI. 将色谱柱连接到HPLCa.色谱柱接头和系统管路注意事项b.测量系统谱带展宽体积和系统方差c.测量梯度延迟（或延迟体积）VII. 其他信息a.使用窄径（3.0 mm内径）色谱柱b.谱带展宽体积对2.1 mm内径色谱柱性能的影响 c.未优化和优化LC/MS/MS系统对比：系统配置更改建议d.沃特世小粒径(2.5 µm)色谱柱 - 快速色谱分离e. XBridge HILIC色谱柱入门f. XBridge Amide色谱柱入门XBridge色谱柱I. 入门指南每根XBridge™色谱柱都附带一份COA报告和一幅性能测试色谱图。
COA报告为XBridge™色谱柱中所用的填料批次所特有，包括批号、未键合颗粒和键合填料分析结果，以及色谱结果和检测条件。
性能测试色谱图提供每根色谱柱的以下信息：批号、色谱柱序列号、USP理论塔板数、USP拖尾因子、保留因子以及色谱条件。
这些数据应妥善保存，以备将来参考。
a. 色谱柱安装注：下述步骤给出的流速适用于粒径5 µm、内径4.6 mm的典型色谱柱。
用户可根据所安装XBridge™色谱柱的内径、柱长、粒径 和柱压相应地提高或降低流速。
赛默飞重磅推出HPLC Troubleshooting APP
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Usually, I recommend storing a column in an organic solvent, if the column is not used for a period of time longer than a weekend.
Paired-Ion Chromatography
Q: Does this apply also to reagents like triethylamine, which are often used to suppress the tailing of basic compounds?
No. These reagents are primarily constituents of mobile phase buffers. They are adsorbed on the packing, but due to the high concentration in which they are typically used, there is little concern about lengthy equilibration. Buffers are commonly used at concentrations around 50 mmol/L, and the concentration of the buffering reagents on the surface of the packing are much lower than the common concentrations of ion-pairing reagents. Therefore, the equilibration of the column with the buffering reagent is much faster than the equilibration with the ion-pairing reagents. Since the issue of long equilibration times does not exist with these simple buffering reagents, columns should be stored in an organic solvent if they will not be used for several days or longer.
Paired-Ion Chromatography
Function of ion pairing reagent
Ion-pairing reagents are used with reversed-phase columns to add ion-exchange properties to the stationary phase. Purely hydrophobic interaction is influenced very little as the ion-pairing reagent is added to then to the problem is to store the column in mobile phase, at least for overnight storage and for storage over weekends.
Paired-Ion Chromatography
Paired-Ion Chromatography
the surface concentration is around 1µ mol/m2
a column may contain around 2 g of packing material, with a specific surface area of 300 m2/g
Paired-Ion Chromatography
If the mobile phase concentration is low, the retention increases in direct proportion to the concentration of the ion-pairing reagent.
The retention times of compounds that interact with the ionpairing reagent depend on its surface concentration
Therefore, one can use the concentration of the ion-pairing reagent to influence the of ionic compounds Ifretention the concentration is high, around10mmol/L, relative to non-interacting compounds. the retention often becomes independent of the This makes it possible to affect thereagent. concentration of the ion-pairing selectivity of a separation. The retention of non-ionic compounds is nearly unaffected by the concentration of the ion-pairing reagent.
Hydrolytic Stability of Reversed-Phase Packings
A pH
At alkaline pH, hydroxyl ions (OH-) can attack and dissolve the silica. The speed of the process depends on the concentration of the hydroxyl ions in the mobile phase, their access to the surface of the silica, and the solubility of the dissolved silica in the mobile phase. In addition, the speed of all these processes is a function of the temperature. What works well at room temperature may represent an unacceptably short column life at 60 º C.
HPLC Troubleshooting Guide
报告人：许维 2013-5-9
Contents
Paired-Ion Chromatography
Hydrolytic Stability of Reversed-Phase Packings
Paired-Ion Chromatography
Q:I am using a chromatographic method on a C18 column that employs an ion-pairing reagent, octyl sulfonic acid. The mobile phase consists of 20% methanol and 80% of the aqueous buffer solution. The aqueous buffer consists of 5mM of the ion pairing reagent and 50 mM acetate buffer; the pH of the aqueous solution is adjusted to pH 4.0 with acetic acid. What bothers me is the lengthy equilibration time necessary to get consistent retention times. What’s wrong?
Tip
Since you are using a buffered mobile phase, the fittings and end-caps of the column should be well tightened to prevent the column from drying out during storage.
Q: This appears to be the problem that I am running into. What is the solution?
It is a good approach for normal reversedphase separations to convert the column to an organic solvent for storage
Contents
Paired-Ion Chromatography
Hydrolytic Stability of Reversed-Phase Packings
Hydrolytic Stability of Reversed-Phase Packings
Q.: Some manufacturers claim that their silica-based reversedIt is columns probably can best be answered bypH your if you phase used to 9 experiments: or 10, while others are happy with the lifetime you able to achieve than to recommend not to use a pH that above 8.are I recently was forced there is nothing wrong with the use9, ofsince a higher than use a reversed-phase column at pH thispH was thethe only manufacturer is recommending. However, I would condition which separated all the compounds of make interest sure that the same separation can obtained on a brandwithout interferences. While this wasbe outside the recommended new column as on a column that has been used for was a while use range of the manufacturer, column life time quite under your If how this is indeed the case, thentake there acceptable. I conditions. now wonder seriously one should the is little reason torecommendation expect trouble. about the pH range over manufacturers’ which their columns should be used.