HPLC Troubleshooting
Contents
1. HPLC Troubleshooting 1 INTRODUCTION Locating and Correcting the Problem Prevention 2 ABNORMAL PRESSURE A No Pressure Reading No Flow B No Pressure Reading Flow Is Normal C Steady High Pressure D Steady Low Pressure E Pressure Climbing F Pressure Dropping to Zero G Pressure Dropping But Not to Zero H Pressure Cycling 3 LEAKS A Leaky Fittings B Leaks at Pump C Injector Leaks D Column Leaks E Detector Leaks 4 PROBLEMS WITH THE CHROMATOGRAM Peak Tailing Peak Fronting Split Peaks Distortion of Larger Peaks Distortion of Early Peaks Tailing Early Peaks More Than Later Ones Increased Tailing as k Increases Acidic or Basic Peaks Tail Extra Peaks Retention Time Drifts Abrupt Retention Time Changes Baseline Drift Baseline Noise Regular Baseline Noise Irregular Broad peaks Loss of Resolution sy O m2 SS os Be a im oo 35 Q All Peaks Too Small R All Peaks Too large 5 PROBLEMS WITH THE INJECTOR A Manual Injector Hard to Turn B Manual Injector Hard to Load C Autoinjector Will Not Turn D Autoinjector Other Problems 6 PROBLEMS DETECTED BY SMELL SIGHT OR SOUND A Solvent Smell B Hot Smell C Abnormal Meter Readings D Warning Lamps E Warning Buzzers F Squeaks and Squeals 7 KEY PROBLEM AREAS AND PREVENTIVE MAINTENANCE A Reservoir B Pump Injector Column Detector General a oa SS 1 INTRODUCTION Locating and Correcting2. Adjust alignment Solution 1 Blockage 2 Jammed mechanism 3 Faulty controller 1 Clear or replace blocked portion 2 See service manual 3 Repair or replace controller 6 PROBLEMS DETECTED BY SMELL SIGHT OR SOUND All senses must be used to identify LC problems Get in the habit of taking a few minutes each day to expose all senses except taste to the LC to get a feel for how the LC performs nor mally This will help to locate problems quickly For example often a leak can be detected by smell before it is seen The majority of problems are identified by sight most of these are included in the proceeding section A Solvent Smell Possible Cause Solution 1 Leak 2 Spill B Hot Smell Possible Cause 1 See Section 3 2 a Check for overflowing waste container b Locate spill and clean up Solution 1 Overheating module 1 a Check for proper ventilation adjust b Check temperature setting adjust c Shut module off see service manual 14 C Abnormal Meter Readings Possible Cause Solution 1 Pressure abnormality 2 Column oven problem 3 Detector lamp failing D Warning Lamps Possible Cause 1 See Section 2 2 a Check settings adjust b See service manual 3 Replace lamp Solution 1 Pressure limit exceeded 2 Other warning lamps E Warning Buzzers Possible Cause 1 a Check for blockage b Check limit setting adjust 2 See
3. b Use guard column c Use precolumn saturator column E Detector Problem Preventive Maintenance 1 Lamp failure decreased detector 1 Replace 6 months or keep spare lamp response increased detector noise 2 Bubbles in cell 2 a Keep cell clean b Use restrictor after cell c Degas mobile phase F General Problem Preventive Maintenance 1 Corrosive abrasive damage 1 Flush buffer from LC and clean when not in use
4. 4 Lower temperature 5 Repair or replace controller Solution 1 See Section C F Pressure Dropping to Zero Possible Cause 1 See Section C Solution 1 See Sections A and B G Pressure Dropping But Not to Zero Possible Cause 1 See Sections A and B Solution 1 See Section D H Pressure Cycling Possible Cause 1 Air in pump 1 See Section D Solution 1 a Degas solvent 2 Faulty check valve s 3 Pump seal failure 4 Insufficient degassing 5 Leak in system 6 Using gradient elution 3 LEAKS b Bleed air from pump 2 Replace check valve s 3 Replace pump seal 4 a Degas solvent b Change degassing methods use Degassex on line degasser 5 Locate leak and correct 6 Pressure cycling is normal due to viscosity changes Leaks are usually stopped by tightening or replacing a fitting Be aware however that over tightened metal compression fittings can leak and plastic fingertights can wear out If a fit ting leak does not stop when the fitting is tightened a little take the fitting apart and inspect for damage e g distorted ferrule or particles on the sealing surface damaged fittings should be discarded and replaced HPLC TROUBLESHOOTING m 5 A Leaky Fittings Possible Cause Solution 1 Loose fitting 1 Tighten 2 Stripped fitting 2 Replace 3 Overtightened fitting 3 a Loosen and retighten b Replace 4 Dirty fitting 4 a Disassemble and clea
5. service manual Solution 1 Solvent leak spill 2 Other warning buzzers F Squeaks and Squeals Possible Cause 1 Locate and correct 2 See service manual Solution 1 Bearing failure 2 Poor lubrication 3 Mechanical wear 1 See service manual 2 Lubricate as necessary 3 See service manual 7 KEY PROBLEM AREAS AND PREVENTIVE MAINTENANCE The chart below lists the most common problems that occur with each LC module In the right hand column are listed preventive maintenance practices that can reduce the failure rate The numbers in parentheses are suggested intervals between maintenance The LC s operator and service manuals may have additional suggestions for preventive maintenance A Reservoir Problem Preventive Maintenance 1 Blocked inlet frit 2 Gas bubbles 1 a Replace 3 6 months b Filter mobile phase 0 5 um filter 2 Degas mobile phase HPLC TROUBLESHOOTING m 15 B Pump Problem Preventive Maintenance 1 Air bubbles 1 Degas mobile phase 2 Pump seal failure 2 Replace 3 months 3 Check valve failure 3 Filter mobile phase use inlet line frit keep spare C Injector Problem Preventive Maintenance 1 Rotor seal wear 1 a Do not overtighten b Filter samples D Column Problem Preventive Maintenance 1 Blocked frit 1 a Filter mobile phase b Filter samples c Use in line filter and or guard column 2 Void at head of column 2 a Avoid mobile phase pH gt 8
6. 1 Contamination on guard or analytical column inlet Normal Problem SPLIT PEAKS 2 Sample solvent incompatible with mobile phase D Distortion of Larger Peaks Possible Cause 1 a Remove guard column and attempt analysis b Replace guard if necessary c If analytical column is obstructed reverse and flush d If problem persists column may be fouled with strongly retained contaminants e Use appropriate restoration procedure f If problem persists inlet is probably plugged g Change frit or replace column 2 Change solvent whenever possible inject samples in mobile phase Solution 1 Sample overload E Distortion of Early Peaks Possible Cause 1 Reduce sample size Solution 1 Wrong injection solvent F Tailing Early Peaks More Than Later Ones Possible Cause 1 a Reduce injection volume b Use weaker injection solvent Solution 1 Extra column effects 1 a Replumb system shorter narrower tubing b Use smaller volume detector cell G Increased Tailing as k Increases Possible Cause Solution 1 Secondary retention effects reversed phase mode 2 Secondary retention effects normal phase mode 3 Secondary retention effects ion pair H Acidic or Basic Peaks Tail Possible Cause 1 a Add triethylamine basic samples b Add acetate acidic samples c Add salt or buffer ionic samples d Try a different column 2 a Add triethylamine basic compo
7. contaminants d Use appropriate restoration procedure if problem persists inlet is probably plugged e Change frit or replace column Solution 1 Detector attentuation too high 2 Detector time constant too large 3 Injection size too small 4 Improper recorder connection R All Peaks Too large Possible Cause 1 Detector attenuation too low 2 Injection size too large 3 Improper recorder connection 5 PROBLEMS WITH THE INJECTOR 1 Reduce attenuation 2 Use smaller time constant 3 Use larger sample loop 4 Use correct connection Solution 1 Use larger attenuation 2 Use smaller sample loop 3 Use correct connection These problems are usually detected while using the injection valve Leaky injection valves are discussed in Section 3 Leaks A Manual Injector Hard to Turn Possible Cause Solution 1 Damaged rotor seal 2 Rotor too tight 1 Rebuild or replace valve 2 Adjust rotor tension HPLC TROUBLESHOOTING 13 B Manual Injector Hard to Load Possible Cause Solution 1 Valve misaligned 2 Blocked loop 3 Dirty syringe 4 Blocked lines C Autoinjector Will Not Turn Possible Cause 1 Adjust alignment 2 Replace loop 3 Clean or replace syringe 4 Clear or replace lines Solution 1 No air pressure or power 2 Rotor too tight 3 Valve misaligned D Autoinjector Other Problems Possible Cause 1 Supply proper pressure power 2 Adjust 3
8. e poorly resolved compounds 11 Column temperature too low 12 Detector time constant too large Normal N 10 11 12 b Check for loose fittings c Check pump for leaks salt build up and unusual noises d Change seals if necessary Adjust settings a Inject smaller column e g 10 ul vs 100 ul or 1 10 and 1 100 dilutions of sample b Reduce response time or use smaller cell c Use as short a piece of 0 007 0 010 inch ID tubing as practical d Reduce response time Increase concentration Replace guard column a Replace column with new one of same type b If new column provides symmetrical peaks flush old column with strong solvent Open inlet end and fill void or replace column Change column type to improve separation Increase temperature do not exceed 75 C unless higher temperatures are acceptable to column manufacturer Use smaller time constant Problem BROAD PEAKS 12 P Loss of Resolution Possible Cause Solution 1 Mobile phase contaminated deteriorated causing retention time to change 2 Obstructed guard or analytical column oth Normal Mo Problem LOSS OF RESOLUTION Q All Peaks Too Small Possible Cause 1 Prepare new mobile phase 2 a Remove guard column and attempt analysis b Replace guard if necessary c If analytical column is obstructed reverse and flush if problem persists column may be fouled with strongly retained
9. een injections or periodi ically during analysis a Reset baseline b Use new mobile phase when dynamic range of detector is exceeded Change wavelength to UV absorbance maximum Problem BASELINE DRIFT 10 M Baseline Noise Regular Possible Cause Solution 1 2 3 4 5 6 Air in mobile phase detector cell or pump Leak He Normal permanar Problem REGULAR BASELINE NOISE Incomplete mobile phase mixing Temperature effect column at high temperature detector unheated Other electronic equipment on same line Pump pulsations N Baseline Noise Irregular Possible Cause 1 a Degas mobile phase b Flush system to remove air from detec tor cell or pump 2 a See Section 3 b Check system for loose fittings c Check pump for leaks salt build up unusual noises d Change pump seals if necessary 3 Mix mobile phase by hand or use less viscous solvent 4 Reduce differential or add head exchanger 5 Isolate LC detector or recorder to deter mine if source of problem is external correct as necessary 6 Incorporate pulse dampener into system Solution 1 O Leak H Normal Mw Problem IRREGULAR BASELINE NOISE Mobile phase contaminated deteriorated or prepared from low quality materials Mobile phase solvents immiscible Detector recorder electronics Air trapped in system Air bubbles in detector Detector cell contaminated
10. en piston Air trapped in pump head Insufficient mobile phase Faulty check valve s Major leak B No Pressure Reading Flow Is Normal Possible Cause b Repair or replace controller Replace piston Degas solvents bleed air from pump prime pump a Replenish reservoir b Replace inlet frit if blocked Replace check valve s Tighten or replace fittings Solution l 2 Faulty meter Faulty pressure transducer C Steady High Pressure Possible Cause 1 Replace meter 2 Replace transducer Solution l 2 WO COND UW A Flow rate set too high Blocked column frit Improper mobile phase precipitated buffer Improper column Injector blockage Column temperature too low Controller malfunction Blocked guard column Blocked in line filter 1 Adjust setting 2 WO OND Ue a Backflush column if permitted b Replace frit c Replace column a Use correct mobile phase b Wash column Use proper column Clear blockage or replace injector Raise temperature Repair or replace controller Remove replace guard column Remove replace in line filter D Steady Low Pressure Possible Cause Solution 1 Flow set too low 2 Leak in system 3 Improper column 4 Column temperature too high 5 Controller malfunction E Pressure Climbing Possible Cause 1 Adjust flow rate 2 Locate leak and correct 3 Use proper column
11. even small amounts of contaminants can cause noise Weak detector lamp Column leaking silica or packing material Mobile phase mixer inadequate or malfunctioning 1 a See Section 3 b Check for loose fittings c Check pump for leaks salt build up unusual noises d Change seals if necessary e Check for detector cell leak 2 Check make up of mobile phase 3 Select and use only miscible solvents 4 a Isolate detector and recorder electroni cally b Refer to instruction manual to correct problem 5 Flush system with strong solvent 6 a Purge detector b Install back pressure device after detector 7 Clean cell by flushing with 1 N HNO never with HCl 8 Replace lamp 9 Replace column 10 Repair or replace the mixer or mix off line if isocratic HPLC TROUBLESHOOTING 11 O Broad Peaks Possible Cause Solution 1 Mobile phase composition changed 1 Prepare new mobile phase 2 Mobile phase flow rate too low 2 Adjust flow rate 3 Leaks especially between column and 3 a See Section 3 detector 4 Detector settings incorrect 5 Extra column effects a Column overloaded b Detector response time or cell volume too large c Tubing between column and detector too long or ID too large d Recorder response time too high 6 Buffer concentration too low Guard column contaminated worn out 8 Column contaminated worn out low plate number N 9 Void at column inlet 10 Peak represents two or mor
12. ift usually to higher absorbance rather than cyclic pattern from temper ature fluctuation Contaminant or air buildup in detector cell Plugged outlet line after detector high pressure cracks cell window producing noisy baseline Mobile phase mixing problem or change in flow rate Slow column equilibration especially when changing mobile phase Mobile phase contaminated deteriorated or prepared from low quality materials Strongly retained materials in sample high k can elute as very broad peaks and appear to be a problem Mobile phase recycled but detector not adjusted 10 Detector UV not set at absorbance maximum but at slope of curve Normal temperature b Use heat exchanger before detector a Use HPLC grade solvents high purity salts and additives b Degas mobile phase before use c Sparge with helium during use a Flush cell with methanol or other strong solvent b If necessary clean cell with 1 N HNO never with HCl a Unplug or replace line b Refer to detector manual to replace window a Correct composition flow rate b To avoid routinely monitor composition and flow rate a Flush with intermediate strength solvent b Run 10 20 column volumes of new mobile phase before analysis a Check make up of mobile phase b Use highest grade chemicals and HPLC solvents a Use guard column b If necessary flush column with strong solvent betw
13. n b Replace 5 Mismatched parts B Leaks at Pump Possible Cause 1 Loose check valves 2 Loose fittings Mixer seal failure Oo Pump seal failure Pressure transducer failure Pulse damper failure Proportioning valve failure ND UW 8 Purge valve C Injector Leaks Possible Cause 5 Use all parts from same brand Solution 1 a Tighten check valve do not over tighten b Replace check valve Tighten fittings do not overtighten 3 a Replace mixer seal b Replace mixer 4 Repair or replace 5 Repair or replace 6 Replace pulse damper 7 a Check diaphragms replace if leaky b Check for fitting damage replace a Tighten valve b Replace purge valve N oo Solution 1 Rotor seal failure 2 Blocked loop 3 Loose injection port seal 4 Improper syringe needle diameter 5 Waste line siphoning 6 Waste line blockage D Column Leaks Possible Cause 1 Rebuild or replace injector 2 Replace loop 3 Adjust 4 Use correct syringe 5 Keep waste line above surface waste 6 Replace waste line Solution 1 Loose endfitting 2 Column packing in ferrule 3 Improper frit thickness 1 Tighten endfitting 2 Disassemble rinse ferrule reassemble 3 Use proper frit see Frit selection guide chart E Detector Leaks Possible Cause Solution 1 Cell gasket failure 1 a Prevent excessive backpressure b Replace gasket 2 Cracked cell window s 2 Replace
14. the Problem A systematic approach is best to identify any problems when troubleshooting the high per formance liquid chromatography HPLC system This guide is organized into five major cat egories of symptoms to help quickly identify the source of the problem s e Pressure abnormalities e Leaks e Problems with the chromatogram e Injector problems e Other problems detected by smell sight and sound When the problem has been corrected record the incident in the HPLC system record book to help with future problems Prevention Many liquid chromatography LC problems can be prevented with routine maintenance For example replacing pump seals at regular intervals should eliminate pump seal failure and its associated problems Section 7 lists the most common problem areas for each LC module as well as preventive maintenance practices to reduce their frequency These sugges tions can be modified to fit any particular model of LC and should be made a regular part of the laboratory routine HPLC TROUBLESHOOTING 3 2 ABNORMAL PRESSURE A change in the operating pressure is a sign that there may be a problem Choose the catego ry below that best fits the symptoms and follow the suggestions to correct the problem A No Pressure Reading No Flow Possible Cause Solution 1 Power off 1 Turn on power 2 Fuse blown 2 Replace fuse 3 Controller setting or failure 3 a Verify proper setting on Oo o N Brok
15. unds b Add acetic acid 3 Add triethylamine basic samples Solution 1 Inadequate buffering Extra Peaks Possible Cause 1 a Use 50 100 mm buffer concentration b Use buffer with pKa equal to pH of mobile phase Solution 1 Other components in sample 1 Normal 2 Late eluting peak from previous injection 2 a Increase run time or gradient slope 3 Vacancy or ghost peaks J Retention Time Drifts Possible Cause b Increase flow rate 3 a Check purity of mobile phase b Use mobile phase as injection solvent c Reduce injection volume Solution 1 Poor temperature control 2 Mobile phase changing 3 Poor column equilibration K Abrupt Retention Time Changes Possible Cause 1 Flow rate change 2 Air bubble in pump 3 Improper mobile phase 1 Thermostat column 2 Prevent change evaporation reaction etc 3 Allow more time for column equili bration between runs Solution 1 Reset flow rate 2 Bleed air from pump 3 a Replace with proper mobile phase b Set proper mobile phase mixture on controller HPLC TROUBLESHOOTING m 9 L Baseline Drift Possible Cause Solution 1 Column temperature fluctuation even 1 a Control column and mobile phase small changes cause cyclic baseline rise and fall most often affects refractive index and conductivity detectors or UV detectors at high sensitivity or in direct photometric mode Nonhomogeneous mobile phase dr
16. window s 3 Leaky fittings 3 Tighten or replace 4 Blocked waste line 4 Replace waste line 5 Blocked flow cell 5 Rebuild or replace FRIT SELECTION GUIDE When Particle Size of Frit Pore Size should be material is 3 4um Qo 0 5um 5 20um Q 4 PROBLEMS WITH THE CHROMATOGRAM 2um Many problems in an LC system show up as changes in the chromatogram Some of these can be solved by changes in the equipment however others require modification of the assay pro cedure Selecting the proper column type and mobile phase are keys to good chromatography A Peak Tailing Possible Cause Solution 1 Blocked frit 1 a Reverse flush column if allowed b Replace inlet frit c Replace column 2 Column void 2 Fill void 3 Interfering peak 3 a Use longer column b Change mobile phase and or column selectivity 4 Wrong mobile phase pH 4 a Adjust pH b For basic compounds a lower pH usually provides more symmetric peaks 5 Sample reacting with active sites 5 a Add ion pair reagent or volatile basic modifier b Change column Normal Problem PEAK TAILING HPLC TROUBLESHOOTING 7 B Peak Fronting Possible Cause Solution 1 Low temperature 2 Wrong sample solvent 3 Sample overload 4 Bad column C Split Peaks Possible Cause 1 Increase column temperature 2 Use mobile phase for injection solvent 3 Decrease sample concentration 4 See A 1 and A 2 Solution
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