hplc trouble shooting - final.ppt

hplc trouble shooting

1. Tips and Tricks of HPLC System
Troubleshooting
By: Dr.Kuldeep Patel

2. Introduction
Thispresentationisintendedasanaidintroubleshooting
problemsthatoccurinthedaytodayrunningofyourHPLC
system.Itisalsointendedasaguidetobestpracticeinsystems
thatarefreefromproblems.Itsprimaryaimsaretoprovidea
logicalprocesswhentroubleshooting,tomaximizesystem
operationtimeandtopromotegoodchromatographicpractices.

3. Troubleshooting Strategy
Any troubleshooting strategy involves five steps:-
1. Identification of the problem.
2. Awareness of the cause (s) of the problem.
3. Isolation of the exact cause of the problem.
4. Rectifying the problem if able.
5.Referringtheproblemtoyourmaintenancemanager.

4. Troubleshooting Processes
The systematic approach should follow a logical sequence, so
that the exact cause of the problem can be found.
1. Gather the facts –not theories.
2. Check the simplest things first –it’s easier.
3.Comparetheperformanceobtainedtotheexpected
performance.
4. List possible causes.
5.Workthroughthepossiblecausesinastep-by-stepmanner
checkingtheoutcomefromanychangesmade.
6.Asalastresort–gethelpfromelsewhere,forexampleyour
instrumentsupplierhelpdeskoryourlocaltechnicalsupport
department.

5. •Itisimportanttorememberthatoncetheproblemisdefined
andpossiblecorrectiveactionisidentified,onlyone
changeatatimeshouldbemade;aftereachchange,the
wholeshouldbecheckedagaintodeterminewhetherthe
problemstillexistsorwhetherthechangecorrectedthe
problem.
•Allproblemsandcorrectiveactionshouldbedocumented
intheinstrumentlogbook.

6. Troubleshooting Isolation & Corrective
Action
•Beforestartinganytroubleshooting,whetheritis
relatedtoinstrumentsorcolumns,itisessentialthat
safelaboratorypracticesbeobserved.
•TherearemanyareasinaHPLCinstrumentthatcan
giverisetosystemandchromatographicproblems.

7. 1.No Peaks/Very Small
Peaks

8. Probable Cause
1.Detectorlampoff.
2.Loose/brokenwirebetween
detectorandintegratoror
recorder.
3.Nomobilephaseflow.
4.Nosample/deteriorated
sample/wrongsample.
5.Settingstoohighondetector
orrecorder.
Remedy
1.Turnlampon.
2.Checkelectricalconnectionsandcables.
3.See“NoFlow”.
4.Besureautomaticsamplervialshave
sufficientliquidandnoairbubblesin
thesample.Evaluatesystem
performancewithfreshstandardto
confirmsampleassourceofproblem.
5.Checkattenuationorgainsettings.
Checklampstatus.Auto-zeroif
necessary.

9. 2. No Flow

10. Probable Causes
1. Pump off.
2. Flow interrupted/
obstructed.
3. Leak.
4. Air trapped in pump
head. (Revealed by
pressure fluctuations.)
Remedy
1.Startpump.
2.Checkmobilephaselevelinreservoir(s).Checkflow
throughoutsystem.Examinesampleloopfor
obstructionorairlock.Makesuremobilephase
componentsaremiscibleandmobilephaseis
properlydegassed.
3.Checksystemforloosefittings.Checkpumpforleaks,
saltbuildup,unusualnoises.Changepumpsealsif
necessary.
4.Disconnecttubingatguardcolumn(ifpresent)or
analyticalcolumninlet.Checkforflow.Purgepump
athighflowrate(e.g.,5-10mL/min),primesystemif
necessary.(Primeeachpumpheadseparately.)If
systemhascheckvalve,loosenvalvetoallowairto
escape.Ifproblempersists,flushsystemwith100%
methanolorisopropanol.Ifproblemstillpersists,
contactsystemmanufacturer.

11. 3.No Pressure/Pressure Lower
than Usual

12. Probable Cause
1.Leak.
2. Mobile phase flow
interrupted/obstructed.
3. Air trapped in pump head.
(Revealed by pressure
fluctuations.)
4. Leak at column inlet end
fitting.
5. Air trapped elsewhere in
system.
Remedy
1.Checksystemforloosefittings.Checkpumpforleaks,salt
buildup,unusualnoises.Changepumpsealsifnecessary.
2.Checkmobilephaselevelinreservoir(s).Checkflow
throughoutsystem.Examinesampleloopforobstructionor
airlock.Makesuremobilephasecomponentsaremiscible
andmobilephaseisproperlydegassed.
3.Disconnecttubingatguardcolumn(ifpresent)oranalytical
columninlet.Checkforflow.Purgepumpathighflowrate
(e.g.,10mL/min),primesystemifnecessary.(Primeeach
pumpheadseparately.)Ifsystemhascheckvalve,loosen
valvetoallowairtoescape.
4.Reconnectcolumnandpumpsolventatdoubletheflowrate.
Ifpressureisstilllow,checkforleaksatinletfittingor
columnendfitting.
5.Disconnectguardandanalyticalcolumnandpurgesystem.
Reconnectcolumn(s).Ifproblempersists,flushsystemwith
100%methanolorisopropanol.

13. 4. Pressure Higher than Usual

14. 1.Probleminpump,
injector,in-linefilter,
ortubing.
2.Obstructedguardcolumn
oranalyticalcolumn
Remedy
1.Removeguardcolumnandanalyticalcolumn
fromsystem.Replacewithunionsand0.010''ID
orlargertubingtoreconnectinjectortodetector.
Runpumpat2-5mL/min.Ifpressureisminimal,
seeCause2.Ifnot,isolatecauseby
systematicallyeliminatingsystemcomponents,
startingwithdetector,thenin-linefilter,and
workingbacktopump.Replacefilterinpumpif
present.
2.Removeguardcolumn(ifpresent)andcheck
pressure.Replaceguardcolumnifnecessary.If
problempersists,columnmaybecloggedwith
stronglyretainedcontaminants.Useappropriate
restorationprocedure.Ifproblemstillpersists,
changeinletfritorreplacecolumn.
Probable Cause

15. 5. Variable Retention Times

16. Probable Cause
1.Leak.
2.Changeinmobilephase
composition.(Smallchangescan
leadtolargechangesinretention
times.)
3.Airtrappedinpump.(Retention
timesincreaseanddecreaseat
randomtimes.)
4.Columntemperaturefluctuations
(especiallyevidentinion
exchangesystems).
Remedy
1.Checksystemforloosefittings.
Checkpumpforleaks,saltbuildup,
unusualnoises.Changepumpseals
ifnecessary.
2.Checkmake-upofmobilephase.If
mobilephaseismachinemixed
usingproportioningvalues,hand
mixandsupplyfromonereservoir.
3.Purgeairfrompumpheadorcheck
valves.Changepumpsealsif
necessary.Besuremobilephaseis
degassed.
4.Usereliablecolumnoven.(Note:
highercolumntemperaturesincrease
columnefficiency.Foroptimum
results,heateluantbefore
introducingitontocolumn.)

17. Probable Cause
5.Columnoverloading.(Retention
timesusuallydecreaseasmassof
soluteinjectedoncolumnexceeds
columncapacity.)
6.Samplesolventincompatiblewith
mobilephase.
7.Columnproblem.(Notacommon
causeoferraticretention.Asa
columnages,retentiontimes
graduallydecrease.)
5.Injectsmallervolume(e.g.,10μL
vs.100μL)orinjectthesame
volumeafter1:10or1:100
dilutionsofsample.
6.Adjustsolvent.Wheneverpossible,
injectsamplesinmobilephase.
7.Substitutenewcolumnofsame
typetoconfirmcolumnascause.
Discardoldcolumnifrestoration
proceduresfail.
Remedy

18. 6. Loss of Resolution

19. Probable Cause
1.Mobilephasecontaminated/
deteriorated(causingretention
timesand/orselectivityto
change).
2.Obstructedguardoranalytical
column
Remedy
1.Preparefreshmobilephase.
2.Removeguardcolumn(ifpresent)and
attemptanalysis.Replaceguard
columnifnecessary.Ifanalytical
columnisobstructed,reverseand
flush.Ifproblempersists,column
maybecloggedwithstrongly
retainedcontaminants. Use
appropriaterestorationprocedure.If
problemstillpersists,changeinletfrit
orreplacecolumn.

20. 7. Split Peaks

21. Probable Cause
1.Contamination on guard or
analytical column inlet.
2. Partially blocked frit.
Remedy
1.Removeguardcolumn(ifpresent)and
attemptanalysis.Replaceguardifnecessary.
Ifanalyticalcolumnisobstructed,reverse
andflush.Ifproblempersists,columnmay
becloggedwithstronglyretained
contaminants.Useappropriaterestoration
procedure.Ifproblemstillpersists,inletfrit
isprobably(partially)plugged.Changefrit
orreplacecolumn.
2.Replacefrit.

22. 8. Peaks Tail on Initial and
Later Injections

23. Probable Cause
1.Samplereactingwithactive
sites.
2.WrongmobilephasepH.
3.Wrongcolumntype.
4.Wronginjectionsolvent.
Remedy
1.Firstcheckcolumnperformancewith
standardcolumntestmixture.If
resultsfortestmixaregood,addion
pairreagentorcompetingbaseoracid
modifier.
2.AdjustpH.Forbasiccompounds,
lowerpHusuallyprovidesmore
symmetricpeaks.
3.Tryanothercolumntype(e.g.
deactivatedcolumnforbasic
compounds).
4.Peakscantailwhensampleisinjected
instrongersolventthanmobilephase.
Dissolvesampleinmobilephase.

24. 9. Tailing Peaks

25. Probable Cause
1.Guard or analytical column
contaminated/ worn out.
2.WrongmobilephasepH.
3.Wrongcolumntype.
4.Wronginjectionsolvent.
Remedy
1.Removeguardcolumn(ifpresent)
andattemptanalysis.Replace
guardcolumnifnecessary.If
analyticalcolumnissourceof
problem,use appropriate
restorationprocedure.Ifproblem
persists,replacecolumn.
2.AdjustpH.Forbasiccompounds,
lowerpHusuallyprovidesmore
symmetricpeaks.
3.Tryanothercolumntype(e.g.
deactivatedcolumnforbasic
compounds).
4.Peakscantailwhensampleis
injectedinstrongersolventthan
mobilephase.Dissolvesamplein
mobilephase.

26. 10. Fronting Peaks

27. Probable Cause
1.Columnoverloaded.
2.Sample solvent
incompatiblewith
mobilephase.
3.Shoulderorgradual
baselinerisebefore
amainpeakmay
beanothersample
component.
Remedy
1.Injectsmallervolume(e.g.,10μLvs.100μL).
Dilutethesample1:10or1:100foldincase
ofmassoverload.
2.Adjustsolvent.Wheneverpossible,dissolve
samplesinmobilephase.
3.Increaseefficiencyorchangeselectivityof
systemtoimproveresolution.Tryanother
columntypeifnecessary(e.g.,switchfrom
nonpolarC18topolarcyanophase).

28. 11. Rounded Peaks

29. Probable Cause
1.Columnoverloaded.
2.Sample-columninteraction.
Remedy
1.Injectsmallervolume(e.g.,10μLvs.
100μL)or1:10and1:10dilutionof
sample.
2.Changebufferstrength,pH,ormobile
phasecomposition.Ifnecessary,raise
columntemperatureorchangecolumn
type.(Analysisofsolutestructuremay
helppredictinteraction.)

30. 12. Baseline Drift

31. Probable Cause
1.Columntemperaturefluctuation.
2.Nonhomogeneousmobilephase.(Drift
usuallytohigherabsorbance,rather
thancyclicpatternfromtemperature
fluctuation.)
3.Contaminantorairbuildupindetector
cell.
4.Mobilephasemixingproblemorchange
inflowrate.
Remedy
1.Controlcolumnandmobilephase
temperature.
2.UseHPLCgradesolvents,highpurity
salts,andadditives.Degasmobile
phasebeforeuse.
3.Flushcellwithmethanolorother
strongsolvent.Ifnecessary,clean
cellwith1NHNO3(neverwith
HCl.)
4.Correctcomposition/flowrate.To
avoidproblem,routinelymonitor
compositionandflowrate
.

32. 13. Baseline Noise (regular)

33. Probable Cause
1.Airinmobilephase,detector
cell,orpump.
2.Incompletemobilephase
mixing.
3.Temperatureeffect(columnat
hightemperature,detector
unheated).
4.Otherelectronicequipmenton
sameline.
5.Leak.
Remedy
1.Degasmobilephase.Flushsystemto
removeairfromdetectorcellorpump.
2.Mixmobilephasebyhandoruseless
viscoussolvent.
3.Reducedifferentialoraddheatexchanger.
4.IsolateLC,detector,recordertodetermine
ifsourceofproblemisexternal.Correct
asnecessary.
5.Checksystemforloosefittings.Check
pumpforleaks,saltbuildup,unusual
noises.Changepumpsealsifnecessary.

34. 14. Baseline Noise
(irregular)

35. Probable Cause
1.Leak.
2.Mobile phase contaminated,
deteriorated, or prepared from low
quality materials.
3.Air trapped in system.
4.Air bubbles in detector.
5.Detector cell contaminated
6. Weak detector lamp.
7. Column leaking silica or packing
material.
Remedy
1.Check system for loose fittings. Check
pump for leaks, salt buildup, unusual
noises. Change pump seals if necessary.
2.Check make-up of mobile phase.
3.Flush system with strong solvent.
4.Purge detector. Install backpressure
regulator after detector. Check the
instrument manual, particularly for RI
detectors (excessive backpressure can
cause the flow cell to crack).
5.Clean cell.
6.Replace lamp.
7.Replace column and clean system.

36. 15. Broad Peaks

37. Probable Cause
1.Mobilephasecompositionchanged.
2Mobilephaseflowratetoolow.
3.Leak(especiallybetweencolumnand
detector).
4.Detectorsettingsincorrect.
5.Extra-columneffects:
a.Columnoverloaded
b.Detectorresponsetimeorcell
volumetoolarge.
c.Tubingbetweencolumnand
detectortoolongorIDtoolarge.
Remedy
1.Preparenewmobilephase.
2.Adjustflowrate.
3.Checksystemforloosefittings.Check
pumpforleaks,saltbuildup,andunusual
noises.Changepumpsealsifnecessary.
4.Adjustsettings.
5.a.Injectsmallervolume(e.g.,10μLvs.
100μL)or1:10and1:100dilutionsof
sample.
b.Reduceresponsetimeorusesmaller
cell.
c.Useasshortapieceof0.007-0.010"ID
tubingaspractical.

38. 18. Negative Peaks

39. Probable Cause
1.Refractiveindexofsolutelessthan
thatofmobilephase(RIdetector).
2.Samplesolventandmobilephase
differgreatlyincomposition
(vacancypeaks).
3.Mobilephasemoreabsorptivethan
samplecomponentstoUV
wavelength.
Remedy
1.Usemobilephasewithlower
refractiveindex,orreverserecorder
leads.
2.Adjustorchangesamplesolvent.
Dilutesampleinmobilephase
wheneverpossible.
3.a.Changepolaritywhenusing
indirectUVdetection,or
b.ChangeUVwavelengthoruse
mobilephasethatdoesnotadsorb
chosenwavelength

40. 19: Ghost Peak

41. Probable Cause
1.Contaminationininjectoror
column.
2.Lateelutingpeak(usuallybroad)
presentinsample.
Remedy
1.Flushinjectorbetweenanalyses
(agoodroutinepractice).If
necessary,runstrongsolvent
throughcolumntoremovelate
eluters.Includefinalwashstep
ingradientanalyses,toremove
stronglyretainedcompounds.
2.a.Checksamplepreparation.
b.Include(step)gradientto
quicklyelutecomponent.