Guide to Successful Operation of Your LC System.pdf

8/9/2019 Guide to Successful Operation of Your LC System.pdf

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Guide to SuccessfulOperation of Your LC System

34 Maple Street

Milford, MA 01757WAT022378TP, Revision 0


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Table of Contents 3

How to Use This Guide ...................................................................... 9

Chapter 1Introduction to System Troubleshooting ........................................... 13

1.1 Get the Facts.......................................................................... 141.2 Check Simple Things First ..................................................... 161.3 Compare System Performance to Established

Benchmarks .......................................................................... 171.4 Identify Possible Causes....................................................... 19

1.5 Use a Systematic Troubleshooting Approach ....................... 211.6 Getting Help .......................................................................... 221.7 Troubleshooting References.................................................. 22

Chapter 2Troubleshooting an LC System ........................................................ 23

2.1 Initial Survey of System Problems ........................................ 252.2 Isolating System Problems ................................................... 29

2.2.1 System Pressure ....................................................... 292.2.2 Baseline Noise........................................................... 39

2.2.3 Changes in Chromatographic Resolution orResults ...................................................................... 67

Chapter 3Troubleshooting System Components ........................................... 109

3.1 Pump Troubleshooting ........................................................ 1103.1.1 Pump Troubleshooting Table.................................... 111

Table of Contents


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4 Table of Contents

3.1.2 Isolating a Defective Pump Head(Modified Ramp Test) .............................................. 120

3.2 Manual Injector Troubleshooting ......................................... 1223.3 Autoinjector Troubleshooting............................................... 1253.4 Column Troubleshooting ..................................................... 1373.5 Detector Troubleshooting .................................................... 1393.6 Data-Handling Device Troubleshooting............................... 151

Chapter 4Good Chromatography/ Operating Practices ................................. 165

4.1 Mobile Phase Preparation and Use .................................... 1654.1.1 Mobile Phase Preparation ....................................... 1664.1.2 Solvent Degassing................................................... 1694.1.3 Solvent Use ............................................................. 1704.1.4 Solvent Changeover Practices................................. 173

4.2 System Plumbing ................................................................ 174

4.2.1 Tubing Connection Practices ................................... 1754.2.2 Cutting Tubing.......................................................... 179

4.3 Chromatographic Performance Tests.................................. 1824.3.1 Measuring Resolution.............................................. 1834.3.2 Measuring Capacity Factor (k') ................................ 1864.3.3 Measuring Selectivity............................................... 1884.3.4 Measuring Column Efficiency (N) ............................ 190

4.4 Measuring System Bandspreading ..................................... 200

Appendix AReference Information .................................................................... 202

A.1 Solvent Properties .............................................................. 202


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Table of Contents 5

A.1.1 Solvent Properties Table ......................................... 203A.1.2 Using Miscibility Numbers (M-Numbers)................. 205

A.2 Refractive Index of Common Solvents................................ 206A.3 Solvent UV Cutoffs ............................................................. 207A.4 Wavelength Selection for Chromophore Detection............. 210A.5 Column Backpressure ........................................................ 212


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1-1 The System Troubleshooting Process ........................................... 151-2 System Map Example.................................................................... 17

2-1 Pressure Release Points within a Typical LC System.................... 312-2 High System Pressure Troubleshooting......................................... 322-3 Low System Pressure Troubleshooting.......................................... 352-4 Erratic System Pressure Troubleshooting...................................... 382-5 Isolating the Source of Changing Chromatographic

Resolution/Results......................................................................... 68

4-1 Comparison of Water Stored in Glass and PlasticContainers ................................................................................... 1674-2 Typical Tubing/Compression Fitting Connection .......................... 1764-3 Adapter Assembly for Incompatible Fittings................................. 1774-4 Cutting Tubing with a Circular Cutter ........................................... 1804-5 Cutting Tubing with a Sharp Object ............................................. 180

4-6 Breaking Stainless Steel Tubing .................................................. 1814-7 Using a Polymeric Tubing Cutter.................................................. 1814-8 Calculating Resolution................................................................. 1834-9 Chemical Factors that Affect System Resolution ......................... 1854-10 Calculating Capacity Factor......................................................... 1874-11 Calculating Selectivity.................................................................. 1894-12 Examples of Low and High Efficiency Peaks............................... 1914-13 Methods of Calculating Column Efficiency .................................. 1924-14 5 Sigma Method Calculation........................................................ 1934-15 4 Sigma Method Calculation....................................................... 1944-16 Tangent Method Calculation ........................................................ 195

4-17 3 Sigma Method Calculation........................................................ 1964-18 1/2 Height Method Calculation..................................................... 196

List of Figures


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4-19 2 Sigma (Inflection) Method Calculation ..................................... 1974-20 Asymmetry-Based Method Calculation ....................................... 198

4-21 Example of Calculating Column Efficiency (N) ............................ 199

A-1 Viscosity for Aqueous Solvent Mixtures....................................... 213


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Table of Contents 8

2-1 System Problems .......................................................................... 252-2 Baseline Noise Summary ............................................................. 412-3 Fluid Path-Related Baseline Noise Troubleshooting ..................... 452-4 Detector Electronics-Related Baseline Noise

Troubleshooting ............................................................................. 60

2-5 Incorrect/Changing Retention Time Troubleshooting .................... 702-6 Abnormal Peak Shape Summary ................................................. 812-7 Abnormal Peak Shape Troubleshooting ........................................ 872-8 Incorrect Qualitative/Quantitative Results Troubleshooting ......... 101

3-1 Pump Troubleshooting ................................................................ 1113-2 Pressure Recording Examples for Modified Ramp Test ............. 1213-3 Manual Injector Troubleshooting ................................................. 1233-4 Autoinjector Troubleshooting ....................................................... 1253-5 Column Troubleshooting ............................................................. 1383-6 Detector Troubleshooting ............................................................ 140

3-7 Computer Hardware Troubleshooting ......................................... 1523-8 Integrator Hardware Troubleshooting .......................................... 1583-9 Chart Recorder Troubleshooting ................................................. 161

A-1 Physical Properties of Solvents .................................................. 203A-2 Refractive Indices of Common Solvents ..................................... 206A-3 UV Cutoffs for Chromatographic Solvents ................................. 207A-4 Wavelength Cutoffs for Common Mobile Phases ....................... 209A-5 Electronic Absorption Bands for Representative

Chromophores ............................................................................ 210

List of Tables


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How To Use This Guide 9

How to Use This GuidePurpose

This guide is an aid in identifying problems within your liquid chromatographic system andhelping to return your system to a proper level of performance. Your system may be acombination of Waters and non-Waters instrumentation.

The primary intent of this guide is to:

Provide a logical approach to troubleshooting an LC system

Outline good chromatography/operating practices

Maximize system operation time

This guide includes a separate quick reference chart that synopsizes the systemtroubleshooting information.

AudienceThis guide is intended for use by a wide variety of LC system operators, whose experiencemay range from novice to expert.


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10 How to Use This Guide

Structure of This Document

This guide is divided into our chapters and a series of Appendixes. Each page includes a

footer, providing easy access to information within the chapter.The table below describes the material covered in each chapter and Appendix.

Chapter 1, Introduction toSystem Troubleshooting

Includes a general discussion on thesystem troubleshooting process.

Chapter 2, Troubleshooting anLC System

Divides the system troubleshooting processinto primary LC system problems:

System pressure (high, no or low,erratic)

Baseline noise Changes in chromatographic resolution

or results, including:

Incorrect retention time

Abnormal peak shape

Incorrect qualitative/quantitative results

Loss of resolution

Possible symptoms, causes, and correctiveactions included for each topic.

Chapter 3, TroubleshootingSystem Components

Troubleshooting tables for individual hardwarecomponents in an LC system:

Pump Manual injector Autoinjector Column Detector Data-handling device

Possible symptoms, causes, and correctiveactions included for each topic.


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How To Use This Guide 11

Related Documents

Refer to the appropriate instrumentation and column operator's manuals and servicemanuals for specific troubleshooting information.

Related Adobe Acrobat Reader DocumentationFor detailed information about using the Adobe Acrobat Reader, refer to the AdobeAcrobat Reader Online Guide . This Online Guide covers procedures such as viewing,navigating and printing electronic documentation from Adobe Acrobat Reader.

Printing From This Electronic Document

Adobe Acrobat Reader lets you easily print pages, pages ranges, or the entire electronicdocument by selecting Print from the File menu. For optimum print quantity, Watersrecommends that you specify a Postscript printer driver for your printer. Ideally, use aprinter that supports 600 dpi print resolution.

Conventions Used in This Guide

This guide uses the following conventions to make text easier to understand.

Purple Text indicates user action. For example:Press 0, then press Enter for the remaining fields.

Italic text denotes new or important words, and is also used for emphasis. Forexample:

An instrument method tells the software how to acquire data.

Chapter 4, GoodChromatography/ OperatingPractices

Proper care and use of a system, intended asa set of operator preventative actions:

Solvent preparation and use System plumbing Chromatographic performance tests

(resolution, k, , and N) Measuring System bandspreading

Appendix A, ReferenceInformation

Reference tables


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12 How to Use This Guide

Underlined, Blue Color text indicates hypertext cross-references to a specificchapter, section, subsection, or sidehead. Clicking this topic using the hand symbolautomatically brings you to this topic within the electronic document. Right-clicking

and selecting Go Back from the popup context menu brings you back to theoriginating topic. For example:

To begin troubleshooting, refer to Chapter 1.2, Check Simple Things First .

Notes, Attentions, and Cautions

Notes call out information that is important to the operator. For example:

Note: Record your results before you proceed to the next step.

Attentions provide information about preventing possible damage to the system orequipment. For example:

Attention: To avoid damaging the detector flow cell, do not touch the flow cellwindow.

Cautions provide information essential to the safety of the operator. For example:

Caution: To avoid chemical or electrical hazards, always observe safe laboratorypractices when operating the system.

Caution: To avoid the possibility of electrical shock and possible injury, always turnoff the detector and unplug the power cord before performing maintenanceprocedures.

Caution: To avoid the possibility of burns, turn off the lamp at least 30 minutesbefore removing it for replacement or adjustment.

STOP


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1

13

1Introduction to SystemTroubleshooting

Troubleshooting a LC system can be a frustrating and sometimes mysterious process. Has

one of these happened your lab? You left your autoinjector running samples over the weekend. When you return on

Monday, you realize that your results gradually progressed from highly reproducibleresults to no peaks in the chromatogram.

The retention times of sample peaks have suddenly shifted within the past hour.

What do you do now? Where do you start?

This chapter outlines a common-sense process for troubleshooting an LC system. Thetroubleshooting information in this chapter is presented with the following assumptions:

Your analysis method has already been developed

Proper system benchmarks have been recorded in the normal operation log

System or instrument performance has degraded as compared with previouslyestablished system benchmarks (see Section 1.3, Compare System Performance toEstablished Benchmarks )

Troubleshooting Stragety

A troubleshooting strategy includes five primary processes:

Identifying the symptom(s).

Understanding possible causes of the symptom. Is it hardware, software, operation,or chemistry related?

Isolating the exact possible causes of the problem. Determining if you have the expertise and items to correct the problem?

Resolving the problem and resuming operation or contacting your local CustomerSupport Representative.


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14 Introduction to System Troubleshooting

1

Troubleshooting process

To implement this strategy, you need a systematic troubleshooting process that works withall types of problems. This process is to:

1. Get the facts.

2. Check simple things first.

3. Compare system performance to established benchmarks.

4. Indentify possible causes.

5. Use a systematic troubleshooting approach.

6. Getting help.

The flowchart in Figure 1-1 illustrates the troubleshooting process. The remainder of thischapter discusses each phase of the strategy.

1.1 Get the Facts

Before troubleshooting, be sure to get all the facts. The fact gathering stage of systemtroubleshooting is an important first step in determining how the system is malfunctioning.

Gather general facts

When you initially believe there is a problem with the performance of your LC system, stepback and consider:

What makes you think something is malfunctioning? What is the evidence (shiftedpeaks, no peaks, baseline drift)?

Is anything different now with the system as compared with establishedperformance (as documented in a log)?

Gather important facts

After this initial gathering process, write down answers to the following questions in your

log: Could someone have changed something in the system (such as detector

sensitivity)?

Are you asking the system to do something that may be beyond its capabilities?

Has the problem ever occurred before?

Is the problem reproducible?

Does this problem occur at any particular time of the day or when anotherinstrument is turned on and off?


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1

Figure 1-1 The System Troubleshooting Process

Your LC SystemExhibits a Problem

NoDid you gather all ofthe facts about the

problem?

Did you perform a visualinspection of the

system?

Did you compare present

system performance withestablished

performance?

Did you indentify allproblem(s) per the exhibited

symptom(s)?

Did you perform asystematic troubleshooting

process?

Can you resolve theproblem?

Problem resolved

Review(Section 1.1 )

No Review(Section 1.2 )




Yes

Yes

Yes

Yes

Yes

Yes



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1.2 Check Simple Things First

When you are certain that something is affecting system performance, always check thesimple things. Finding the easy solution saves time and frustration.

Attention: Always observe safe laboratory practices when troubleshooting. Know thechemical and physical properties of the solvents. Refer to the Material Safety Data Sheetfor the solvents in use.

Attention: To avoid electric shock, power down the instrument and disconnect thepower cord before removing the cover and examining the instrument.

Look for obvious "clues"

Make a visual inspection of the system, investigating:

Alarms triggered on any components?

Fluid leaks?

Pump pressure reading normal? Power cord inserted in both the outlet and the instrument rear panel?

Power switch on?

Fuses blown?

Sufficient solvent in the reservoir?

Electrical cables connected between devices? Are connections correct?

Instrument settings changed or incorrectly set?

Solvent flowing out of detector waste tubing?

Correct column?

If the visual inspection does not uncover anything obvious, compare current systemperformance to established system operation (see Section 1.3, Compare SystemPerformance to Established Benchmarks ).

STOP

STOP


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Compare System Performance to Established Benchmarks 17

1.3 Compare System Performance to EstablishedBenchmarks

To help you identify normal operation conditions:

Record a map of your LC system

Keep a log

Run test chromatograms regularly

These three practices allow you to compare present system operation with establishedsystem performance.

Note: A log is especially important in a lab where the system is shared by multipleoperators.

Recording a System Map

When your LC system is initially installed, sketch a general map of the fluidic and electricalconfiguration. Label the individual connections. Use this map to review these fluidic andelectrical connections and reconfigure when necessary. Figure 1-2 is an example of asystem map.

Figure 1-2 System Map Example

Pump

Pump/ system

controller

Auto-injector

ComputerA/D

interface

Detector

In-Linefilter

Column

INJ GND + -1 V

OUT

IEEE-488 CONNECTIONRS-232 CONNECTION

Chan 11 V analog out signal

(from detector)

Inject startsignal

RS-232connection

(225 l pump heads) (250 l sample loop) Waste

IEEE-488 connectionIEEE-488

connection

Controlcable

Start int.

Electrical cable

Res A Res B

Fluid line- 1/16 inch (1.6 mm) O.D.,0.020 inch (0.5 mm) I.D.

stainless steel tubing (3 ft)

Fluid line- 1/16 inch ( 1.6 mm) O.D.,0.060 inch (1.5 mm) I.D. teflon

tubing

Fluid line- 1/16 inch (1.6 mm) O.D.,0.009 inch (0.23 mm) I.D.

stainless steel tubing (kept to a minimum)

Fluid line- 1/16 inch ( 1.6 mm) O.D.,0.009 inch (0.23 mm) I.D. stainlesssteel tubing (1 ft) (30 cm)

(C18 3.9 mm X 150 mm)

Red line

CONTROL CABLE

FLUID CONNECTION

ELECTRICAL SIGNALS

--

Blue line


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1

Keeping a log

Record operating conditions (pressure, flow rate, and so on) in a logbook. When you haveproblems with your system, use the logbook to compare system information, such as:

Instrument settings

Parts and/or components recently replaced (with manufacturer serial number anddate that the change was made)

Maintenance procedures (what and when)

Number of samples run (system throughput) in your method

Sample, standard, and mobile phase information for each method used

Test chromatogram, including specific operating conditions (column, flow rate,mobile phase, backpressure, and so on) for each column used

Method table(s) used for your application(s) (such as gradient conditions, integrationparameters)

Running a Test Chromatogram

Always run a test chromatogram when: A new system is installed

An instrument is replaced or added

A column is replaced or added

New mobile phase is prepared

When running the standard, establish a set of standard conditions. Collect the data andrecord:

Pressure

Resolution (Rs) of critical peak pairs

Capacity factor (k') for each peak

Selectivity ( ) Column efficiency (N)

Refer to Chapter 4, Good Chromatography/ Operating Practices for information oncalculating and troubleshooting resolution, capacity factor, selectivity, and columnefficiency.

Keep the test chromatogram and associated data in your logbook. When you believe thereis a problem, rerun the standard. Compare the results to establish:

If the test results are not significantly different from those previously recorded, theproblem is method-specific.


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1

Identify Possible Causes 19

If the test results are different, either the column or an instrument have changed.Repeat the test with a new (or known good) column. If the results of the secondcolumn are satisfactory, the problem is with the first column. If both columns fail, the

problem is most likely with an instrument.

1.4 Identify Possible Causes

Use the exhibited symptoms to narrow down the possible causes within the system. Toidentify a problem(s):

Identify all of the symptoms Match the symptoms with the potential possible causes

Identifying the Symptoms

Perform a survey of your system to determine where the exhibited symptom(s) mayoriginate.

Some symptoms are:

Variable or unusual system pressure (high, no or low, erratic)

Noisy or drifting baseline

Incorrect or changing retention time(s)

Abnormal peak shapes (such as broad, tailing, or fronting peaks)

Incorrect qualitative/quantitative results (too many peaks, too few peaks, wrong

answer)


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Identifying the Possible Causes

From the isolated symptom(s), write down a list of suspected possible causes in yourlogbook.

For example:

The troubleshooting tables in Chapter 2, Troubleshooting an LC System , and Chapter 3,Troubleshooting System Components are examples of possible causes for a particularsymptom.

Isolating the Possible Cause

From your list of possible causes, isolate the problem areas within your LC system. Thisnarrows down whether the problem is hardware, software, operation, or chemistry related.You can then follow a systematic troubleshooting approach to resolve the problem (seeSection 1.5 ).

Symptom Possible Causes

Sudden high systembackpressure

Blocked column frit Sample precipitation on column

Changed or incorrect flow rate Blocked tubing

Defective pump pressuretransducer

Drop in operating temperature


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Use a Systematic Troubleshooting Approach 21

1.5 Use a Systematic Troubleshooting Approach

Once all of the possible causes are identified, resolve the problem using a systematicapproach:

Follow a logical sequence to correct the problem

Make only one change at a time to the system, starting with the easiest changes

Document the results of each change

Following a Logical Sequence

After listing the possible causes of the problem, follow a logical troubleshooting sequenceto narrow down the exact source of the problem.

For example, if the symptom is unusually high backpressure, loosen each fitting, startingat the detector waste outlet, and observe if pressure drops. Continue to work toward thepump until the pressure drops, identifying the possible cause of the problem.

Note: For detailed information on system troubleshooting, refer to Chapter 2,Troubleshooting an LC System .

Making Only One Change at a Time

During troubleshooting, make only one change to the system at a time to the system untilthe problem is resolved. Parts of the system you may make changes to include:

Instrument settings

Column Sample

Operating conditions

Mobile phase

Replacement of suspected malfunctioning parts

Replacement of suspected malfunctioning instruments

Note: If the replacement part does not correct the problem, be sure to put back theoriginal part. Conversely, rebuild or discard a malfunctioning part/assembly.

Making only one change at a time allows you to understand the affects on systemperformance.


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Documenting Changes

Always take notes as you make changes to the system, such as changing settings,swapping component parts, or swapping an instrument. Record-keeping is a valuable partof the systematic approach because it provides you with:

A trail back to configuring the system in its original form if your troubleshooting pathis incorrect

A trail back if new problems or symptoms arise

If you proceed through the system troubleshooting process and the identical symptom(s)persist, reevaluate your diagnosis and investigate another area in the system.

1.6 Getting Help

It is important that you feel confident with your troubleshooting and maintenance abilities.If you feel that the problem is beyond your area of expertise, consult someone within yourlab with more experience. If that does not help resolve the problem, call the CustomerSupport Department (or service specialist) for help.

Follow the troubleshooting process outlined in this guide to narrow down the symptom tothe most likely problem source. With that knowledge, you are able to concretely discussyour diagnosis with the Customer Support representative, saving valuable time andconfusion.

1.7 Troubleshooting ReferencesFor additional information on liquid chromatography system troubleshooting or liquidchromatography in general, refer to the following reference material:

The operator's manuals and service manuals for your LC instrumentation andcolumns

Dolan, John, W., and Snyder, Lloyd, R. (1989), Troubleshooting LC Systems , TheHumana Press Inc., P.O. Box 2148, Clifton, NJ, 07015

Snyder, L.R. and Kirkland, J.J., Introduction to Modern Liquid Chromatography ,Second Edition, Wiley-Interscience, New York, 1979

The "LC Troubleshooting" column in LC/GC Magazine


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2

2Troubleshooting an LCSystem

This chapter outlines LC system troubleshooting. It divides system troubleshooting into:

Initial survey of system problems Isolating possible causes of system problems, including:

System backpressure (high, no or low, erratic)

Baseline noise

Changes in chromatographic resolution or results, including:

Incorrect/changing retention times

No peaks or abnormal peak shapes

Incorrect qualitative/quantitative results

Loss of resolution

Troubleshooting information in this chapter is presented with the assumption:

Your analysis method has already been developed

Proper system benchmarks have been recorded in the normal operation log System or instrument performance has degraded as compared with previously

established system benchmarks (see Section 1.3 )

For background information on the system troubleshooting process, refer to Chapter 1,Introduction to System Troubleshooting .

For specific instrument and column troubleshooting, refer to Chapter 3, Troubleshooting

System Components . When troubleshooting, keep the following safety considerations inmind:

Attention: Always observe safe laboratory practices when troubleshooting. Know thechemical and physical properties of the solvents. Refer to the Material Safety Data Sheetfor the solvents in use.

Attention: To avoid electric shock, power down the instrument and disconnect the powercord before removing the cover and examining the instrument.

STOP

STOP


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24 Troubleshooting an LC System

2

Attention: Ultraviolet light is emitted during UV and fluorescence detector operation. Toprevent eye damage, eye protection must be worn while troubleshooting the detector withits covers removed.

If handling integrated circuit boards, use an anti-static mat and wear an anti-staticwrist strap to remove excess static charge and prevent damage to the board.

Do not touch any of the integrated circuit chips or other components which do notspecifically require manual adjustment.

When You Call

Many problems with your LC system Customer Support can be easily corrected by theprocess outlined in this chapter. If you cannot correct a condition for a Waters product,contact one of the following:

Your Waters service specialist

Waters Customer Support Department

Waters Service offices (listed on the rear cover of this guide)

To expedite service, have the following information available when you call:

1. Completed Normal Operation Log and test sample chromatogram for method

2. Nature of symptom(s)

3. Type and model number of pump (single or multiple solvent)

4. Flow rate

5. Operating pressure

6. Mobile phase(s)

7. Type and model number of injector (manual or autoinjector)

8. Type and model number of detector (UV, RI, fluorescence, conductivity,electrochemical)

9. Detector settings (wavelength, sensitivity, and so on)

10. Type and serial number of column

11. Sample matrix and components

12. Data system

13. Software version (if applicable)

STOP


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Initial Survey of System Problems 25

2

2.1 Initial Survey of System Problems

When a problem occurs, first perform a visual check of the system. Look for leaks,disconnected tubing, disconnected cables, changed instrument settings, and so on.Review your system map System map (as described in Section 1.3, Compare SystemPerformance to Established Benchmarks ) to verify all fluidic and electrical connections.

Table 2-1 is a summary of the most common problems affecting system operation.

If you determine one (or more) of these are the problem, refer to Chapter 3,Troubleshooting System Components , or the instrument/column operator's manual for thecorrective action.

Table 2-1 System Problems

Component Areas to Check

General System Instrument not plugged in/not turned on No power at wall outlet Blown power fuse Incorrect instrument settings Cooling fans in instrument not running Leaks

Incorrect air or gas supplies Disconnected or improper electrical cabling Too many instruments on same circuit Incorrect grounding between instruments


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Pump/SolventFlow

Incorrect flow rate setting Solvent reservoir(s) empty

Solvent not degassed or sparged

Obstruction or crimp in tubing

Improper tubing ID

Improperly cut tubing Incorrect or worn ferrules/compression screws

Plugged in-line filter

Plugged solvent inlet filter

Air in pump inlet lines or pump heads

Leaking plunger seal

Leaking fittings Malfunctioning inlet/outlet check valve

Pressure transducer out of calibration

Solvent reservoir positioned lower than pumpinlet

Manual Injector Obstruction in sample loop

Insufficient injection volume Inconsistent injection volume

Incorrect or damaged syringe

Leaking vent tube or valve

Wrong sample loop

Inconsistent injection sequence

Leaking or worn seals

Contaminated syringe

Injector out of adjustment

Table 2-1 System Problems (Continued)



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Initial Survey of System Problems 27

2

Autoinjector Air in syringe Insufficient sample in vial Improper sample vial position Incorrect or improper settings Leaking or worn seals Valve failure No or low air pressure Wrong syringe size

Detector Defective source lamp Incorrect or improper settings (sensitivity,

attenuation, wavelength, time constant)

Insufficient time for lamp to stabilize Auto zero left enabled Incorrect optical filter and/or lamp Dirty flow cell Air bubble in flow cell Dirty reference electrode Dirty working electrode Incorrect reference and sample solvent

balance (RI detector) Leaking flow cell

Computer Wrong A/D sampling time Improper output voltage signal from detector Improper attenuation, peak width, area reject,

noise rejection parameters Poor peak integration Incorrect calibration or sample table




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Integrator Improper attenuation Improper input voltage range Auto zero left enabled by detector Improper output voltage signal from detector Improperly inserted paper

Chart recorder Improper chart speed Improper input voltage range Improper output signal from detector Gain improperly adjusted Reversed polarity Auto zero left enabled on detector

Auto zero left enabled on chart recorderChromatography Operating environment

Column not equilibrated Mobile phase problem (improperly degassed,

prepared, or filtered; incorrect for application;contaminated)

Sample problem (improperly prepared orfiltered; insoluble with mobile phase;degraded)

Contaminated column Obstruction in guard column or column inlet

frit Improper column or precolumn




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Isolating System Problems 29

2

2.2 Isolating System Problems

This section covers the most common symptoms exhibited during operation, including:

Note: If a specific instrument or the column in your system exhibits a symptom notaddressed in this section, refer to Chapter 3, Troubleshooting System Components , forinstrumentation and column troubleshooting.

2.2.1 System PressureTroubleshooting overview

This section covers troubleshooting:

High system pressure

Low system pressure Erratic (fluctuating) system pressure

System pressure troubleshooting is presented in a flow diagram format. Use Figure 2-2 through Figure 2-4 to investigate pressure problem sources.

Exhibited symptom Refer to

System pressure(high, no/low, or erratic

Section 2.2.1

Baseline noise Section 2.2.2

Changes to chromatographic resolutionand results

Incorrect retention time

No peaks or abnormal peaks

Incorrect qualitative/quantitative

results Loss of resolution

Section 2.2.3

Section 2.2.3.1Section 2.2.3.2Section 2.2.3.3

Section 4.3


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System pressure reference point

To identify a pressure change from normal operation, it is critical that you have a pressurereference point. System pressure is affected by the column, flow rate, mobile phase, andtemperature, and can vary greatly with different methods.When running a gradient,fluctuations in system pressure may be due to viscosity changes between solvents.

Each time you install a new column or start a new method, equilibrate the system andrecord the system pressure (both with and without the column in line) to use as acomparison. Use the normal operation log to record system pressure.

Gradual versus sudden pressure increase

When high system pressure occurs, it is important to note whether the pressure increasewas gradual or sudden.This can help you isolate the problem source.

If the pressure has risen gradually (over a series of injections), it may be due to:

Particulates in the sample or the mobile phase which have accumulated in thein-line filter or column frits

Debris from failed fluid seals

If the pressure has risen suddenly , it may be due to:

Particulates in one sample

A system hardware problem (such as blocked tubing)

Collapse of the column packed bed

High pressure locations within an LC system

To isolate the origin of high system pressure, loosen inlet or outlet fittings as instructed inFigure 2-2 and observe if pressure stays the same or reduces.

Attention: Always observe safe laboratory practices when troubleshooting. Always wearsafety glasses and gloves. Know the chemical and physical properties of the solvents.Refer to the Material Safety Data Sheet for the solvents in use.

Figure 2-1 presents pressure release points in a typical LC system. Use Figure 2-1 as areference when reviewing the high pressure troubleshooting flow diagram in Figure 2-2 .

To relieve pressure, slowly loosen the fitting. Use a tissue to prevent solvent spray and alsoto collect any spilled solvent.

STOP


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Attention: f the source of high pressure is within the detector flow cell, use extremecaution when relieving high pressure buildup. Many detectors (especially RI andfluorescence) have fragile flow cells. Before backflushing the detector to remove the

blockage, review the flow rate specifications and backpressure limits for that flow cell in thedetector operator's manual.

Figure 2-1 Pressure Release Points within a Typical LC System

STOP

Single pumpgradient system

Solvent reservoirsA, B, C, D

Gradientmixer

(optional)

n ec or(manual or

autoinjector)Outlet

OutletInlet

OutletInlet

Column

Detector

Wastereservoir

In-linefilter

or guardcolumn

(optional)

Outlet Inlet

Outlet Inlet

Multiple pump

Inlet

Outlet

OutletInlet

Gradientmixer

(optional)

Pump A Outlet

Solvent reservoirA

Solvent reservoirB

Pump B

OutletPumpOption 2

Option 1


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Figure 2-2 High System Pressure Troubleshooting

System pressureproblem?

Are highly viscoussolvents being used?

YesHas ambienttemperature changed?

Is pump flow rate set

too high?

Figure 2-2 continuedon next page.

Check solvent viscosity (seeAppendix A, Reference

Information ). The observedpressure may be normal for this

column and solvent blend. Ifnecessary, change to a less

viscous solvent.

Set to correct flow rate.

Check the operating temperature.If temperature is too low, stabilize

operating environment.

High, no/low, orerratic?

Go toFigure 2-3

Go toFigure 2-4

Yes

Yes

No

No

Erratic

High

No/Low

No


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Figure 2-2 High System Pressure Troubleshooting (Continued)

Yes

No

Is pressuretransducer operating

correctly?

Loosen fitting atdetector waste outlet.

Pressure reduced?

Loosen fitting at

detector inlet.Pressure reduced?

Figure 2-2 continuedfrom previous page.

Loosen fitting atcolumn inlet.

Pressure reduced

Loosen fitting at guard

column or in-line filter outlet.Pressure reduced?

Loosen fitting atcolumn outlet.

Pressure reduced?

Figure 2-2 continuedon next page.

Loosen fitting at

transduceroutlet. Set flow

rate to zero.Does pressurefall to zero? (Ifyes, retighten

fitting).

Adjusttransducer. Can

pressure bezeroed?

Defectivetransducer.Replace per

pump operatorsmanual.

Blocked outlet connectingtubing. Clean or replace tubing

per Chapter 4 and detectoroperators manual.

Blocked fluid path (flow cell).

Clean blockages per detectoroperators manual.

Blocked outlet connecting tubing.Clean or replace tubing per

Chapter 4 and columnoperators manual.

Blocked or voided column.Clean column per column

operators manual. If problempersists, replace the column.

Blocked outlet connecting tubing.Clean or replace tubing perChapter 4 and column/filter

operators manual.

Yes

Yes

Yes

Yes

No

No

No

Yes

No No No

No

Yes Yes


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Figure 2-2 High System Pressure Troubleshooting (Continued)

Yes

No

Loosen fitting at guardcolumn or in-line filter

inlet. Pressure reduced?

Loosen fitting atinjector outlet.

Pressure reduced?

Loosen fitting at mixerinlet. Pressure

reduced?

Loosen fitting at pumpoutlet. Pressure

reduced?

Loosen fitting atinjector inlet. Pressure

reduced?

Pump problem, callCustomer Support.

Blocked in-line filter or guardcolumn. Replace components.

Blocked outlet connectingtubing. Clean or replace tubing

per Chapter 4 and injectoroperators manual.

Blockage within injector orconnecting tubing. Clean

blockages per injector operatorsmanual. Verify sample solubility.

Blockage within connecting tubingor mixer. Clean or replace tubing

per Chapter 4 . If mixer problem,see operators manual.

Blocked outlet connecting tubing.Clean or replace tubing per

Chapter 4 and pump operatorsmanual. Verify solvent miscibility.

Yes

Yes

Yes

Yes

No

No

No

Figure 2-2 continuedfrom previous page.

No


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Figure 2-3 Low System Pressure Troubleshooting

No or low systempressure

Figure 2-3 continuedon next page

Check fuse, replace ifnecessary.

Yes

NoIs pump fuse blown?

Turn pump on.

Yes

NoIs pump on (are fansrunning)?

Is there solvent in thereservoir?

Yes

NoIs there solvent flowingfrom the detector

outlet?Refill reservoir.

Is the low pressure limitset to a value higher

than operating pressure?

Can solvent be drawneasily from draw-off

valve?

Has the pump beenproperly primed?

Set correctpressure limit.

Plugged in-line filter.Clean filter. Replace if

necessary.

Prime the pump perpump operators

manual.

Yes

Yes

No

Yes

No

Yes

No

No



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Figure 2-3 Low System Pressure Troubleshooting (Continued)

Figure 2-3 continued

from previous page


Is there air in thesolvent inlet lines?

Are both pump headsworking? Place finger onindicator rod (if present)

to check.

Set correct flow rate.

No

YesHas flow rate been set

too low?

Yes

Figure 2-3 continued

from previous page

Remove air from eachinlet line.

Pump problem. Referto Section 3.1, Pump

Troubleshooting .

Check operatingtemperature. If too high,stabilize environment.

No

YesHas ambienttemperature changed?

Tighten any loosefittings (not overtight).

No

YesAre there any visibleleaks?

Verify solvent used. Ifnecessary, change

solvent.

Yes

NoIs correct solvent

being used?


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Figure 2-3 Low System Pressure Troubleshooting (Continued)

Figure 2-3 continuedfrom previous page

Call CustomerSupport

Autoinjector problem.Refer to autoinjectoroperators manual.

No

YesIs autoinjector stuck inpurge?

Measure flow rate atdetector outlet with a

graduated cylinder anda stop watch. Is solventflow rate much differentthan the pump setting?Yes

NoIs the correct flow rateactually being deliveredthrough fluid system?

Pump problem. Referto Section 3.1, Pump

Troubleshooting .


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Figure 2-4 Erratic System Pressure Troubleshooting

Yes

Is pressure transduceroperating properly?

Has the pump beenproperly primed?

Erratic systempressure.

Call CustomerSupport.

Set flow rate tozero and checktransducer. Is

pressure stable atzero flow?

Adjust transducer.Can pressure be

zeroed?

Defectivetransducer.Replace per

pump operatorsmanual.

Prime the pump per thepump operators manual. If

pressure erratic, refer toSection 3.1, PumpTroubleshooting .

No

Yes

No No No

Yes

Do pressure changesreproducibly follow the

gradient change?

No

Are you using volatilesolvents (such as

ethers)?

Pump problem. Refer toSection 3.1, PumpTroubleshooting .

Check solvent miscibility.Change solvents (see

Appendix A, ReferenceInformation ).

Yes

No

Yes

No

Yes

No

No

Degas/helium sparge

solvent(s). See Chapter 4 .

Degas/sparge solvents (seeChapter 4 ).

Are miscible solventsbeing used?

Is the mobile phaseproperly sparged and

degassed?

Are both pump headsworking? Place finger on

indicator rod (ifappropriate) to verify.

Are you running agradient?

Pressure fluctuations arenormal due to viscosity

changes between solvents.

No


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2.2.2 Baseline NoiseTroubleshooting overview

This section covers troubleshooting baseline noise. It assumes baseline noise is eitherfluid path-related (mobile phase, pump, column) or detector electronics- related.

Baseline noise is characterized as:

Non-cyclic (erratic) baseline noise

Cycling (short or long-term) baseline noise

Baseline drift

Noise spikes on the baseline

Table 2-3 covers fluid path-related noise and Table 2-4 covers detector electronics-relatednoise.

Note: Data-handling device electronics noise is covered in Section 3.6 .

Table 2-2 summarizes the baseline noise symptoms used in Table 2-3 andTable 2-4 . From Table 2-2 :

1. Review the baseline noise symptoms and select the one that best typifies yoursystem problem.

2. Proceed to the page number of the baseline noise symptom (in Table 2-3 orTable 2-4 ).

3. Review the list of possible causes and follow the corrective actions.

Isolating the source of baseline noise

To isolate the source of the baseline noise:

1. Turn off your pump to stop solvent flow to the system.

2. Monitor the baseline for a few minutes. Note the following:

If there is significant improvement in the baseline, the problem is within the fluidpath (pump/mobile phase/flow path/column). Refer to Table 2-3 .

Note: Some flow sensitive detectors, such as RI and electrochemical, mayrequire a significant time to stabilize once flow stops.

If the noise continues, the problem is within the detector or its electricalconnections. Proceed to step 3.

3. Disconnect the detector electrical cables from the data-handling device (A/Dinterface to the computer, computer, integrator, or chart recorder).


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4. Attach a jumper source to the input terminals on the data-handling device (suchas a wire or paperclip).

If the noise continues, the problem is within the data-handling device. Refer to

Section 3.6, Data-Handling Device Troubleshooting . If the noise stops, the problem is within the detector or its electrical

connections. Refer to Table 2-4 .

Baseline noise summary

Table 2-2 is a summary of the baseline noise symptoms listed in Table 2-3 and Table 2-4 . These baseline noise examples were generated using the following operating conditions:

Note: The amplitude of the noise shown in the examples in this section have beengenerated in a lab environment. The signal amplitude can vary depending upon theseverity of the problem.

Parameter Setting

Pressure 1000 psi

Detector absorbanceunits

1 AU

Recorder sensitivity 10 mV full-scale

Chart speed 1 cm/min

Output signal fromdetector

10 mV


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Table 2-2 Baseline Noise Summary

Baseline Noise Symptom Refer to

Non-cyclic (erratic) noiseFluid path problem: Table 2-3

Detector electronics problem: Table 2-4

Short-term cycling noise

Fluid path problem Table 2-3

Air bubble in flow cell

Unstabilized system

Time

A U o r m

V

Time

A U o r m

V

Leak in detector flow cellTime

A U o r m

V

Defective lamp Operating at high sensitivityTime

A U o r m

V

Time

A U o r m

V

Inadequate solvent blending

Air bubbles indetector flow cell

Time

A U

o r m

V

Time

A U o r m

V

Time

A U o r m

V



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Short-term cycling noise(Continued)

Detector electronics problem:

Table 2-4

Long-term cycling noise

Fluid path problem: Table 2-3


Table 2-2 Baseline Noise Summary (Continued)


Cycling equipment

Time

A U o r m

V

Temperature fluctuations

A U o r m

V

Time


A U o r m

V

Time


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Baseline driftFluid path problem: Table 2-3

Detector electronics problem Table 2-4

Noise spikesFluid path problem: Table 2-3



Contaminated column

A U o r m

V

Time

Insufficient stabilization

Time

A U o r m

V

Air bubbles in fluid path

Time

A U o r m

V


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Defective lamp

Time

A U o r m

V


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Table 2-3 Fluid Path-Related Baseline Noise Troubleshooting

Symptom Possible Cause Corrective Action

Non-cyclic (erratic) noise(originating from fluid path)

Large air bubble trappedin detector flow cell

To remove the air bubble,purge the detector flow cell orapply slight pressure on thedetector waster outlet (seedetector operators manual).Note: To prevent air bubblesfrom forming in the flow cell,add a 1 to 3 foot (30 to 90 cm)length of 0.009 -inch (0.23mm) ID, 1/16 -inch (1.58 mm)ID, 1/16 -inch (1.58 mm) ODtubing to the detector wasteoutlet. This tubing functionsas a flow restrictor to increasebackpressure. A 3 foot (90cm) piece of tubing provides30 to 50 psi (2 to 3 atm) ofbackpressure at 1 mL/min inwater.

Keep in mind thebackpressure limits of the flowcell (with RI, fluorescence,

conductivity, andelectrochemical detectors)before attaching this tubing.

Air bubble trapped inreference electrode(electrochemical detectoronly)

Remove the referenceelectrode and gently shake itto remove the air bubble.Replace the referenceelectrode.

Normal baseline

Air bubble in flow cell

Unstabilized system

Time

A U o r m V

Time

A U o r m V

Time

A U o r m V

Leak in detector flow cellTime

A U o r m V


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Non-cyclic (erratic) noise(originating from fluid path)(Continued)

Small air bubblestraveling through the flowpath

Prime the pump to remove air(see pump operators manual).To prevent additional airbubbles, ensure the mobilephase is properly degassed orhelium sparged (seeChapter 4 for information).

System not stabilized orchemically equilibrated

Allow all system components(such as the column anddetector) sufficient time tostabilize and chemicallyequilibrate. Note the operatingconditions of your application(such as mobile phase,detector settings, detectortype). Refer to the instrumentor column operators manualfor recommended equilibrationtimes.If running an automatedgradient method, ensure

sufficient and reproducibleequilibration times are usedbetween injections.Note: If using ion-pairingreagents, ensure that the firsttime you use the column youprovide a sufficient time andvolume of solvent to

adequately equilibrate thecolumn (for example, runninga total volume of 100 mL of a5 mM solution at 1 ml/min).

Table 2-3 Fluid Path-Related Baseline Noise Troubleshooting (Continued)



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Mobile phasecontaminated

Discard the contaminatedmobile phase and:

Clean the solventreservoir. Clean orreplace the solvent inletfilter. To clean the fil ter,

remove and sonicateusing 6N nitric acid,water (repeat 3 times),followed by methanol.

Prepare and filter freshsolvent using only highquality reagents andHPLC-grade solvents(see Section 4.1 forsolvent preparation anduse considerations)

Flush and re-equilibratethe system.




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Detector flow cell leaking Remove the detector coverand check for leaks. If leaksare not visible, perform thefollowing:1. Flush the detector with a

non-buffered misciblesolvent, followed bymethanol.

2. Attach a nitrogen or heliumsource to the detector inlet.Slowly blow the flow celldry.

3. Monitor the baseline for

noise. If noise disappears,there is a leak within thedetector flow cell.

Repair/replace the detectorflow cell (see detectoroperators manual)




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Column contaminated To verify this problem as apossible cause, replace allcolumns in the system with aunion (or with a known goodcolumn of the same type).Return the mobile phase, andmonitor the baseline.Note: If you require additionalsystem backpressure duringthis verification, use a piece of0.009 -inch (0.23 mm) IDtubing (or suitable restrictor)instead of a union.

If the problem stops, clean or

replace the contaminatedcolumn as outlined in thecolumn operators manual.If the problem continues, thiscould be due to:

Solvent properties suchas miscibility. Refer to

Section 4.1 andAppendix A, ReferenceInformation forinformation.

Contaminated mobilephase (refer to theMobile phasecontaminated PossibleCause listed above forthe corrective action

Contaminated guardcolumn in-line filter(clean or replace asoutlined in the operatorsmanual).




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Short-term cycling noise(seconds to minutes)(originating from fluid path)

Erratic pump pressure/pump pulsations

Refer to Figure 2-4 (inSection 2.2.1 ) to verify andcorrect the source of erraticpressure. If erratic pressurecontinues, refer to Section 3.1,Pump Troubleshooting .



Normal baseline

Time

A U o r m

V


Air bubbles in detectorflow cell

Time

A U o r m

V

Time

A U

o r m

V

Time

A U o r m

V



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Short-term cycling noise(seconds to minutes)(originating from fluid path)

Inadequate solventbending

To confirm a mixing problem:1. With the column in-line,

pump 5-10 columnvolumes of 100% A andmonitor the baseline. Thisprovides both a constant

composition and sufficientvolume to equilibrate thecolumn and reach thedetector flow cell.

2. Pump several pre-mixedsolvents (for example,50/50, 95/5, 70/30, or the

mixture you are running)and monitor the baseline.

If the baseline is acceptablewith 100% A, but becomesnoisier when running mixtures,there is a mixing problem.Verify the mixing problem andcorrect as outlined below:1. Use of immiscible solvents.

Verify miscibility of solvents(see Appendix A) andchange to more misciblesolvents.

2. Malfunction in the pump,the pump solventproportioning valve, or thehigh-pressure mixer.Troubleshoot perSection 3.1, PumpTroubleshooting




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Short-term cycling noise(seconds to minutes)(originating from fluid path)(Continued)

Inadequate solventbending (continued)

3. Inadequate blending afterthe pump. The solution isto add additional mixing.However, the mixingrequired depends on theseverity of the problem.Verify and correct asfollows:

Increase laminar flowmixing and residencetime (the time to passthrough the mixer). Adda 6 to 12 inch (150 to300 mm) length of0.040-inch (1.0 mm) IDtubing between thepump outlet and theinjector. This tubinglength adds only a smallsystem delay. volumeand does not distortgradient shape.

If the tubing does notresolve the problem, adda larger mixer volume fora more vigorous mixing.Add one or more WatersGradient Flow Mixers

between the pump outletand injector. The mixingchamber providesconsistent solventblending with minimalvolume and gradientdistortion.




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Short-term cycling noise(seconds to minutes)(originating from fluid path)(Continued)

Inadequate solventbending (continued)

Note: Ensure that you use amixing chamber with thesmallest possible volume.This prevents introducingadditional delay volume ordistorting gradient shape.

4. Use premixed solvent(s).

Pump Inlet tubing loose,bent, or blocked

Check the tubing. If loose,tighten. If bent, straighten. Ifblocked, replace (seeChapter 4, GoodChromatography/ OperatingPractices ).

Large air bubble indetector flow cell

Refer to the Corrective Actionunder the Non-cyclic (erratic)noise symptom listed above.

Dirty or malfunctioningpump inlet check valve

Refer to Section 3.1, PumpTroubleshooting

Worn pump plunger seal Refer to Section 3.1, PumpTroubleshooting

Solvent from detectoroutlet dripping into wastecontainer (primarilyaffecting flow sensitivedetectors such as RI andelectrochemical)

Position detector outletagainst the side next to thewaste container.




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Long-term cycling noise(minutes to hours)(originating from fluid path)

Ambient temperaturefluctuations

Stabilize operatingenvironment temperature. Ifthe problem continues:

Use a column heater(run 5 Celsius aboveambient).

Relocate the system orcolumn to a thermallystable environment.

Avoid placing thesystem in directsunlight.

Solvent is being recycledfrom detector waste outletback through LC system

Unless absolutely necessary,do not recycle solvent throughyour LC system. Only usefresh and filtered solvent foryour application.



Normal baselineTime

A U o r m

V


A U o r m

V

Time


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Baseline drift(originating from fluid path)(Continued)

Detector flow cell leaking Refer to Corrective Actionunder the Non-cyclic (erratic)noise symptom listed above.

Contaminated column Refer to the Corrective Actionunder the Non-cyclic (erratic)noise symptom listed above.

Solvent is being recycledfrom detector waste outletback through LC system

Unless absolutely necessary,do not recycle solvent throughyour LC system. Only usefresh and filtered solvent foryour application.

Leak(s) in system Check all fitting for leaks. If

there is a leaky fitting, tightenit (do not overtighten).If the leak continues, replacethe fitting and ferrule (seeSection 4.2 forconsiderations).




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Stationary phase bleed To verify this problem, replaceall columns in the system witha union. Rerun the mobilephase and monitor thebaseline.Note: If you require additionalsystem backpressure duringthis verification, use a piece of0.009 -inch (0.23 mm) IDtubing instead of a union.

Ensure your operatingconditions are suitable for thecolumn (for example, solventcompatibility, pH range, and so

on). If the operating conditionsaffect the column: Select a different mobile

phase Select a different

columnRefer to the column operators

manual for information.Incorrect wavelength forsolvent

Verify the backgroundabsorbance of the mobilephase using a spectrometer. Ifthe background is high (youare unable to zero the detectorbaseline), the mobile phase

contains a UV-absorbingcompound, which causesbaseline drift.Operate at a wavelength thatis above the UV cutoff for themobile phase (see Table A-3 )or change the solvent(s).




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Mobile phase contains astabilizer or there is achange in the stabilizer

Use a preservative-freesolvent(s).Note: Separations mayrequire adjustment ifchanging to apreservative-free solvent.

Late-eluting samplecomponent

Wash the column with anappropriate strong solvent.

Unbalanced solvents(gradient operation)

Attempt to balance the UVabsorbance with the mobilephase. If constrained bychemistry, run a gradient blankand subtract the baseline.

Noise spikes(originating from fluid path)

Small air bubblestraveling through the fluidpath

Refer to the Corrective Actionunder the Non-cyclic (erratic)noise symptom listed above.



Normal baselineTime

A U o r m

V

Air bubbles influid path

Time

A U o r m

V


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Noise spikes(originating from fluid path)(Continued)

Pump head cavitation Refer to Section 3.1, PumpTroubleshooting .

Particles in detector flowcell

Clean or backflush thedetector flow cell (asdescribed in the detectoroperators manual).

Improper grounding ofpump or autoinjectorelectrical connections

Use a shielded signal cableand attach the shield to onedevice only .If shielding is not the problem,plug the pump or autoinjector

into another outlet on adifferent electrical circuit, If aseparate outlet is unavailable,use a line power conditioner.




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Table 2-4 Detector Electronics-Related Baseline Noise Troubleshooting


Non-cyclic (erratic) noise(originating from detector)

Detector not stabilized Allow detector lampsufficient time to stabilize(until baseline is stable).Detector equilibration timevaries based on the type ofdetector used and operatingparameters (such as

wavelength, sensitivity,background, potential,and/or current). Refer to thedetector operators manualfor recommendedequilibration times.

Normal baselineTime

A U o r m

V

Defective lamp

Operating at highsensitivity

Time

A U o r m

V

Time

A U o r m

V


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Non-cyclic (erratic) noise(originating from detector)(Continued)

Defective detector lamp Verify lamp energy usingdetector diagnostics. Ifenergy is belowspecification (as comparedwhen the lamp was new),replace the lamp.Note: Some detectorsallow you to adjust lampenergy to compensate fordecreased energy. Refer toyour detector operatorsmanual for information onadjusting lamp energy.

Contaminated detector flowcell

Refer to Section 3.5,Detector Troubleshooting .

Detector electronicsproblem

Detector malfunction.Contact Customer Support.

Cable loose or improperlyconnected betweendetector and data-handling

system (computer,integrator, or chart recorder)

Verify that the correctdetector output signal isproperly connected to the

data-handling device.Ensure that any relatedoutput signal switch settingsare in the proper position.Refer to the detectorsoperators manual anddata-handling deviceoperators manual.

Table 2-4 Detector Electronics-Related Baseline Noise Troubleshooting (Continued)



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Detector improperlygrounded

Use a shielded signal cableand attach the shield to onedevice only .If shielding is not theproblem, plug the detectorinto another outlet on adifferent electrical circuit. Ifa separate outlet isunavailable, use a linepower conditioner.

Cycling equipment or radiofrequency (RF) interference

Isolate the detector fromother equipment in the lab,especially devices withlarge electric motors. Then:

Check circuitgrounding and linevoltage quality (referto the Detectorimproperly groundedPossible cause abovefor the corrective

action). Ensure that the

detector and thedata-handling deviceare on the samecommon ground.Relocate if necessary.

If necessary, relocatethe detector to anarea where RF is nota problem or use aFaraday cage aroundthe detector.




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Data-handling devicegain/sensitivity setting toohigh

Change to a lowergain/sensitivity setting (seedata-handling deviceoperators manual).

Leak from referenceelectrode (electrochemical

detector only)

Repair or replace thereference electrode (see

detector operators manual).

Foulded reference electrode(electrochemical detectoronly)

Renew reference electrodefilling solution and/orreplace the frit (see detectoroperators manual).

Contaminated or scratched

working electrode(electrochemical detectoronly)

Clean or polish working

electrode (see detectoroperators manual). Ifproblem persists, replacethe working electrode.

Short-term cycling noise(originating from detector)(seconds to minutes)


Refer to the CorrectiveAction under the Non-cyclic(erratic) noise symptom

listed above.



Normal baselineTime

A U o r m

V

Cycling equipment

Time

A U o r m

V


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Short-term cycling noise(originating from detector)(seconds to minutes)(Continued)

Internal detectortemperature improperly set(heater cycles on and offtoo frequently)

Correctly set the detectorinternal heater (seedetector operators manual).

Long-term cycling noise(originating from detector)

(minutes to hours)


Stabilize operatingenvironment temperature to

allow full equilibration. Ifproblem continues:

Relocate the detectorto a thermally stableenvironment or closeany open air vents.

Avoid placing the

system in directsunlight.


Refer to the CorrectiveAction under the Non-cyclic(erratic) noise symptomlisted above.



Normal baseline

Time

A U o r m

V


A U o r m

V

Time


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Baseline drift(originating from detector)

Detector not stabilized Refer to the CorrectiveAction under the Non-cyclic(erratic) noise symptomlisted above.

Change in ambient

temperature

Refer to the Corrective

Action under the Long-termcycling noise symptomlisted above.

Contaminated detector flowcell

Refer to Section 3.5,Detector Troubleshooting .

Fouled reference electrode(electrochemical detectoronly)

Renew reference electrodefilling solution (see detectoroperators manual). Ifproblem persists, replacethe frit.



Normal baselineTime

A U o r m

V

Insufficient stabilization

Time

A U o r m

V


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Contaminated or scratchedworking electrode(electrochemical detectoronly)

Clean or polish workingelectrode (see detectoroperators manual). Ifproblem persists, replacethe working electrode.

Noise spikes

(originating from detector)

Defective detector lamp Refer to the Corrective

Action under the Non-cyclic(erratic) noise symptomlisted above.

Cycling equipment or radiofrequency (RF) interference Refer to the CorrectiveAction under the Non-cyclic(erratic) noise symptomlisted above.

Detector electronicsproblem

Detector malfunction.Contact your CustomerSupport Department.



Normal baselineTime

A U o r m

V

Defective lampTime

A U o r m

V


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2.2.3 Changes in Chromatographic Resolution or ResultsChromatographic problems typically exhibit themselves as:

Incorrect or changing retention time(s)

No peaks or abnormal peak shape(s)

Loss of resolution

Incorrect qualitative or quantitative results

When your chromatographic results are unacceptable (as compared with establishedperformance), reinject your application standard(s) and translate your observations into

specific problems with your system.For example, problems that appear to be due to abnormal peak shape can be due to otherissues with chromatographic performance. Before investigating solutions to abnormalpeak shape, verify that incorrect retention time is not affecting your chromatography.

Troubleshooting unacceptable chromatographic results involves two stages:

Sequentially evaluate your chromatographic results (retention time, peak shape,

resolution, and qualitative/quantitative results) Isolate the source of the problem

The flow diagram in Figure 2-5 presents questions to help you narrow down your problem.Once you isolate the problem, refer to the appropriate troubleshooting section.


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Figure 2-5 Isolating the Source of Changing Chromatographic Resolution/Results

Yes

Are retection timescorrect?

Has peak shapechanged?

Are the peaks properlyidentified and theresults correct?

Has resolutionchanged?

Troubleshoot incorrect/changingretention times. Proceed to

Section 2.2.3.1 .

Troubleshoot abnormal peakshapes. Proceed to

Section 2.2.3.2

Troubleshoot resolution. Proceedto Section 4.3 .

Troubleshoot qualitative and/orquantitative results. Proceed to

Section 2.2.3.3

No

Yes

Yes

No

No

No

Changes inchromatographic

resolution or results.

Yes

Chromatographyis acceptable.


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2.2.3.1 Incorrect/Changing Retention TimesTroubleshooting overview

Incorrect retention time is characterized as: Erratic (changing back and forth from run to run)

Steadily increasing

Steadily decreasing

Changed to a constant new value (incorrect but reproducible)

Table 2-5 covers incorrect/changing retention time. From the table:

1. Review the retention time symptoms and select the symptom that best typifies yourproblem.

2. Review the possible causes and follow the corrective actions.

Retention time stability factors

Retention time stability is affected by:

System and column equilibration Mobile phase (composition, preparation, improper degassing, stability)

Column age

Operating temperature

Pump performance (flow rate, pressure, loss of prime)

Isolating retention time changes

When evaluating retention times changes, it is critical that you have previously establishedsystem performance benchmarks. From those benchmarks, you determine whetherretention times:

Erratically change in a different direction (either increasing or decreasing) from runto run.

Change in the same direction (increasing or decreasing) from run to run.

Suddenly change to a new value (increased or decreased), which is outside theallowable range for the assay, and then hold at that value for a number of runs.

T bl 2 5 I /Ch i R i Ti T bl h i


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Table 2-5 Incorrect/Changing Retention Time Troubleshooting


Erratic retention times(changing from run to run)

System not stabilized orchemically equilibrated

Allow all systemcomponents (such as thecolumn and detector)sufficient time to stabilizeand chemically equilibrate.Note the operatingconditions of your

application (such as mobilephase, detector settings,and detector type). Refer tothe instrument or columnoperator's manual forrecommended equilibrationtimes.If running an automatedgradient method, ensuresufficient and reproducibleequilibration times areused between injections.Note: If using Ion-pairingreagents, ensure that thefirst time you use thecolumn you provide asufficient time and volumeof solvent to adequatelyequilibrate the column (forexample, running a totalvolume of 100 mL of a5 mM solution at1 mL/min).

T bl 2 5 I t/Ch i R t ti Ti T bl h ti (C ti d)


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Erratic retention times(changing from run to run)(Continued)

Erratic pumppressure/pump pulsations(due to problems such asair bubbles, worndirty/malfunctioning checkvalves, worn plunger seals,worn plunger)

Refer to Figure 2-4 (inSection 2.2.1 ) to verify thesource of erratic pressure.If erratic pressurecontinues, refer toSection 3.1, PumpTroubleshooting .

Injection volume/sampleconcentration too high(sample overload),disrupting equilibrium

Reduce the injectionvolume or dilute thesample with mobile phase.If using a weaker solvent,you can inject up to 10% ofcolumn void volume. Ifusing a stronger solvent,

you can inject up to 1% ofcolumn void volume.


Stabilize operatingenvironment temperatureto allow full equilibration. Ifproblem continues:

Use a column heater

(run 5 Celsius aboveambient).

Ensure the solvent isstirred.

Relocate the systemor column to athermally stableenvironment or closeany open air vents.

Table 2-5 Incorrect/Changing Retention Time Troubleshooting (Continued)


Table 2 5 Incorrect/Changing Retention Time Troubleshooting (Continued)


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Inadequate solventblending

To confirm a mixingproblem:1. Premix, filter, and degas

the mobile phase.

2. With the column in-line,pump a minimum of

5 to 10 column volumesthrough a single pump(or solvent line) toequilibrate the column.

3. Inject a standard aminimum of 3 times andcompare the

reproducibility of theretention times to theprevious injections (witherratic retention times).

If retention times arereproducible with thepremixed solvent, thisindicates a solventblending problem.Verify the mixing problemand correct as outlinedbelow:1. Use of immiscible

solvents. Verifymiscibility of solvents

(see Appendix A,Reference Information )and change to moremiscible solvents.



Table 2 5 Incorrect/Changing Retention Time Troubleshooting (Continued)


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Inadequate solventblending

2. Malfunction in thepump, the pump solventproportioning valve, orthe high-pressure mixer.Troubleshoot perSection 3.1, PumpTroubleshooting .

3. Inadequate blendingafter the pump. Thesolution is to addadditional mixing.However, the mixingrequired depends onthe severity of theproblem. For theCorrective Action, referto Table 2-3 , the"Short-term cyclingnoise" symptom.

4. Use premixedsolvent(s).





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Column contamination To verify this problem as apossible cause, replace thecolumn with a known goodcolumn of the same type.Rerun the analysis andobserve if retention timesstabilize. If the problem

stops, clean or replace thecolumn as outlined in thecolumn operator's manual.If the retention timecontinues to be erratic, thiscould be due to:

Solvent propertiessuch as miscibility.Refer to Section 4.1 and Appendix A,Reference Information for information.

Contaminated mobilephase (refer to the"Mobile phasecontaminated"Possible Cause laterin this table for thecorrective action).

Contaminated guardcolumn or in-line filter(clean or replace asoutlined in theoperator's manual).

Increased or decreasedretention times(continuously changing inthe same direction)

Pump flow rate changed Verify the solvent flow ratesetting. Set to appropriateflow rate for the application.





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