Troubleshooting Common HPLC Problems

1 Troubleshooting Common hplc ProblemsWaters Corp. 2002 Potential Sources of Chromatographic ProblemszMobile PhasezInjectorzIn-Line FilterzColumnzDetectorzSamplezPumpzGuard ColumnzConnecting Tubing and FittingszIntegrator/RecorderSoftwareScie ntist/Analyst --need for logical approach to save timeWaters Corp. 2002 ProblemCHEMISTRYzCOLUMNzGUARD COLUMNzMOBILE PHASEzSAMPLE/VIALSzPUMPzINJECTORzDETECTO RzDATA COLLECTIONzBAND SPREADING/CONNECTIONSzCOLUMNSzSAMPLE VIALST roubleshooting StrategyTry to simplify --assess impact on lab efficiency --inspect the chromatography --try to categorize troubleshoot the easiest to fix items firstMECHANICALW aters Corp.

2 2002 Detector CellWaters c 1998 How to Categorize --Inspect Chromatogram How do you get sharp peaks with excellentresolution? Well ShapedBands -- Well Separated (Good Mechanical And Chemical Performance)Waters Corp. 2002 Detector CellWhy Do You Get Distorted Peaks? Why do you get alldistorted peaks? DistortedBands Mechanical problem Chemical problem Injector- Too Strong Sample Solvent Voided Column Poor Connections_Waters Corp. 2002 Voids - high back pressure, distorted and/or double peaksAll Peaks Distorted --Mechanical ProblemSimilar DistortionWaters Corp. 2002 All Peaks Distorted Chemical ProblemIncorrect Sample Solvent STRONGER than mobile phase(Too strong)StrongerWaters Corp.

3 2002 Detector CellWhy Do You Get One/Some Distorted Peaks? Why do you get one or somedistorted peaks? DistortedBand Chemical problem Cation exchange of one analyte to particle surfaceWaters Corp. 2002 Great Peak Shape for Some Peaks, but Others Have Poor Peak Shape --- Chemical problem (Not Mechanical)Minutes0510152025 Example of chemical induced tailing for this compound --Wrong choice in packing material brandor mobile phase pHOther peaks have Great Peak shapeWaters Corp. 2002* Extra-Column (Non Column) Band SpreadingzInjection VolumezInjector (seal problem )zConnecting Tubing zInjector to ColumnzColumn to DetectorzEnd-fittings and FritszDetector VolumeMechanical * Column Itself Voiding Plugged frits Remember: the band is initially created by the injector}Waters Corp.

4 " Parker " WatersStyleOther Waters ColumnsWaters Spherisorb andmany other brandsColumn Installation and Connections - MechanicalWaters Corp. 2002zMake sure column connected correctly zMake sure nut and ferrule are seatedInstallation -- MechanicalNOTE: Column connector not seated properly --creates void (Parker ferrule position into a Waters End-fitting)Good Seal(PEEK Slip Connectors Easier to Use)VoidWaters Corp. 2002 Band Spreading due to ImproperColumn Connection- MechanicalColumn ConnectionBand SpreadingDead VolumeProperImproperWaters Corp. ".020".040"Note the differences for the inner diameter of this connecting tubingExtra-Column Band Spreading Tubing ID -- MechanicalWaters Corp.

5 2002 Effect of Connecting Tubing on System Band ".020".040"Samplebanddispersion inside tubingNote: longer tubing lengthscreates more Band SpreadingWaters Corp. 2002 Effect of Band Spreadingon Customer s plumbing Lack of sensitivity Quattro Ultima Metabolite study Improved plumbing Replace 10 connectionsbetween the injector, switching valve and mass spec Removed 4 feet of extra inch id tubing2X Gain in SensitivityMetabolite in tail of peak now resolvedOriginal Customer s SystemOptimized SystemJ. LiWaters Corp. 2002 Now, Perform Plate Count --Install Column (Isocratic) Plate Count Information included in AppendixIf System OK, Now Test Column and System Together (Plate Count)--If poor result --implies Column Failure/ConnectionVRWaters Corp.

6 2002 Performance MonitoringColum n Efficiency: N = the num ber of Theoretical Platesa = is a constant depending on the Method usedtr= retention tim e of p eakW = the peak width (tim e units) at a given peak heightN = a( )trW2 METHOD aPeak W idth at Half Height W idth at Peak Height (5 S igm a) : Value will depend on CONDITIONS.(Analyte k, flow rate, temperature etc.)Waters Corp. 2002 Well packed columnMechanical -- Column Packing Material FlushWaters Corp. 2002 Well packed columnGood Results(System OK , Column OK, Connections OK)Waters Corp. 2002 Column Collapse -- MechanicalVoided column- ChannelingPacking Material SettledPacking Material SettledWaters Corp.

7 2002 Voids - high back pressure, distorted and/or double peaksColumn Collapse (voiding)(shock / high pH {dissolution of particle})All Peaks DistortedWaters Corp. 2002 Broad and Tailing peaks* PARTIALLY PLUGGED COLUMN INLET FILTER- Remove end-fitting* CONTAMINATED IN-LINE FILTER- Replace frit* CONTAMINATED GUARD COLUMN- Replace guard column/insertPeak Shape ProblemsSee Appendix for more informationOther Reasons forPoor Peak ShapeWaters Corp. 2002 Most Common problem in hplc :zDistorted peaks will cause integration or resolution problemsIndication that optimal column performance is not being attainedPeak Shape Problems Waters Corp. 2002 Peak Shape ProblemszColumn DestroyedzIncorrect Sample SolventzSecondary InteractionszColumn Overload Mass Overload Volume OverloadzOther Extra-Column Effects Sampling Rate Time ConstantWaters Corp.

8 2002 Voids - high back pressure, distorted and/or double peaksColumn Collapse (voiding)(shock / high pH {dissolution of particle})All Peaks DistortedWaters Corp. 2002 All Peaks AffectedCOLUMN- Connection- Replace frit/ Guard Column- Regenerate or replace columnCOLUMN ITSELF DESTROYEDpH <2 washes off functional grouppH >8 dissolves silica basefronting peaksPeak Shape Problemsdouble peaksWaters Corp. 2002 Dissolution ofSilica gelpH Limitations of Traditional Silica Based Packing of Bonded LigandXTerra Hybrid -- Wide pH RangeWaters Corp. 2002 Hydrolysis of a Bonded Phase Material: Monofunctional LigandNote: Sometimes called Column Bleed +HCl++Low pH (hydrolysis of ligand)SiCCCCCCCCH3CH3H3 CClOHSiOOOSiCCCCCCCCH3CH3H3 COSiOOOSiCCCCCCCCH3CH3H3 CHOOHSiOOONote: as hydrolysis of ligand continues, you will obtain less retentivity and potentially poor peak shape for phaseWaters Corp.

9 2002 Dissolution ofSilica gelpH Limitations of Traditional Silica Based Packing of Bonded LigandXTerra Hybrid -- Wide pH RangeWaters Corp. 2002 Solubility of Silica in Water (ppm)1 2 3 4 5 6 7 8 9 10240-220-200-180-160-140-120-100-80-60- 40-20-0-pHSilica Solubility CurveSilica pH 2 - 8 Polymer pH 2 -12At pH >8 silica dissolvesMobile Phase pH and Column Life-timepH > 8 causes VOIDINGfor traditional silica/ bonded particlesWaters Corp. 2002 Peak Shape ProblemszColumn DestroyedzIncorrect Sample SolventzSecondary InteractionszColumn Overload Mass Overload Volume OverloadzOther Extra-Column Effects Sampling Rate Time ConstantWaters Corp.

10 2002 All Peaks Distorted Chemical ProblemIncorrect Sample Solvent STRONGER than mobile phase(Too strong)StrongerWaters Corp. 2002 All Peaks Distorted Chemical ProblemIncorrect Sample Solvent STRONGER than mobile phase(Too strong)STRONGER SAMPLE SOLVENTC olumn: SymmetryShield RP18, m, x 150 mmGuard Column: Sentry Guard Column SymmetryShield RP18, 5 mTemperature: 30 CMobile Phase: TFA:Methanol(60:40)Detection: UV at 210 nmFlow Rate: 1 mL/minInj. Volume: 30 L Sample Diphenhydramine (IS)3. in in 80% MeOH, 2% Corp. 2002 Peak Shape ProblemszColumn DestroyedzIncorrect Sample SolventzSecondary InteractionszColumn Overload Mass Overload Volume OverloadzOther Extra-Column Effects Sampling Rate Time ConstantWaters Corp.