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Troubleshooting Common HPLC Problems

1. Lift off Point Moves Earlier Retention times are shorter Dr Shulamit Levin Medtechnica Column Collapse voids high back pressure distorted and or double peaks Column Volume Overload EFFECT OF INJECTION VOLUME ON PEAK DISTORTION Volume Overload Lift off Point Remains Constant Retention times are longer Extra Column Effects Isocratic LC Time Constant Differences Detector setting left is 0 1 secs right is 10 secs note the noisy baseline on left chromatogram Dr Shulamit Levin Medtechnica Contaminated In Line Filter New frit 800 psi Contaminated frit 2500 psi Performance Monitoring Use Your Test Method Known Performance Monitor at least One Peak in one injection Plate Count Peak width relative to RT Peak Response Inject Multiple Runs Precision at least 5 injections Accuracy Use Control Samples Solvent Composition Retention Time Problems Clearly specify HOW the Mobile Phase is to be prepared m Reproducibilit Drifting Retention p y g pe E gt Solvent Composition Equilibration Temperature gt Stationary Phase Stability 60 40 pH Control Column Contamination lon Pairing Hydrophobic Collapse pH Reminder Measure pH Before the organic is added Temperature Control Retention Time Reproducibility Non Column Influences pH e Neutrals No Influence e Acids Reduced Retention with Increasing pH
2. 30mm guard column column coupler Column Protection Sentry Nova Pak Cis Sentry Symmetry Cs Adsorbosphere Cis Upchurch ODS Brownlee NewGuard RP 18 Alltech Econosil Cis Zorbax Reliance Rx Cs 0 20 40 60 80 100 of original efficiency Effect of guard column on HPLC columns efficiencies Analytical column Nova Pak Cis 150 x 3 9mm or 4 6mm except Zorbax Rx C 150 x 4 6mm Sample was 0 5yL injection acenapthene 2 9 mg mL and acetone 34 uL mL in ACN Water Dr Shulamit Levin Medtechnica Column Protection 1 Guard column should be regarded as a cost effective sacrifice to extend analytical column life time 2 Should contain IDENTICAL packing material as the analytical column e g using a different C18 with different retention properties could actually destroy the separation Well designed well packed guard column will actually IMPROVE the analytical separation efficiency 1 Sulfanilamide 2 Sulfadiazine 3 Sulfathiazole 4 Sulfamerazine 5 Sulfamethazine 6 Succinylsulfathiazole Column Protection Conditions Column Symmetry C 3 9 mm X 150 mm with Sentry Guard Column 3 9 mm X 20 mm Mobile Phase water methanol glacial acetic acid 79 20 1 2 Injection 5020 E Start 0 4 6 8 0 Minutes Chromatogram of Life time Test Guard Column Changed Every 500 Injections 12 Column Protection B After 550 injections on same Seniry guard column New Sentry Guard co
3. Filter solvents and samples Sample too VISCUOUS SIPHONING RI CONDUCTIVITY ECD Increase system volume MIXING PROBLEMS SPIKES BUBBLES Degas solvent CHEMICAL COMPOUNDS ELUTING OFF COLUMN Run strong solvent until baseline is stable SOLVENTS IN GRADIENT ARE NOT PURE Change the solvent batch or manufacturer Check if the solvents are grandient grade POOR ELECTRICAL CONNECTION LOOSE WIRING Clean and tighten detector leads check wiring replace spade lugs LAMP RELAY TRYING TO FIRE A DEAD LAMP Replace lamp ELECTRICAL NOISE Change circuits remove source Common sources include switching valves compressors muffle furnaces fraction collectors power conditioners lighting poor power source Dr Shulamit Levin Medtechnica ai UT O TFT CYCLIC BASELINE TEMPERATURE FLUCTUATIONS Thermally insulate Move away from ventilation Increase cell temperature MIXING PROBLEMS Increase system volume GAS IN MOBILE PHASE Degas solvents ELECTRICAL PROBLEMS Change circuits remove source ERRATIC PUMP Repair pump PLUG Remove obstruction flush system NO PEAKS INSTRUMENTAL CHEMICAL Injector not making injections e Column retaining all compounds Pump not pumping e Bad or wrong mobile phase Dead detector e Bad or wrong standard or sample Integrator recorder not wired e Wrong guard column correctly Gain setting too low WHAT TO DO Leaks Remove column and inject acetone solution to make
4. Good Seal s m NOTE column inlet connector not seated properly PEEK Connectors Easier to Use THF makes PEEK brittle Extra Column Band Spreading Tubing Contribution 009 040 020 note the differences of the inner diameter of this tubing Dr Shulamit Levin Medtechnica Extra Column Band Spreading WA Pe H Wa N i ma una BIN Column Connection Contribution Performance Monitoring Effect of Connecting Tubing on System Bandspreading 009 020 040 J OSN Diluted Distorted Band sample band dispersion inside tubing Measuring The Instruments Contribution Perform An Instrument Band Spreading Test Performance Monitoring Using 5 sigma efficiency method measure the peak width at 4 4 of peak height Convert to microliters using the following equation ee m a 100 ut where 1min 20cm chart speed 1 mL min flowrate 1000 pL mL volume correction factor Typical LC System should be 100uL 30uL Microbore System should be no greater than 20uL Dr Shulamit Levin Medtechnica Performance Monitoring To perform a measurement disconnect column from system connect injector directly to detector Parameter Setting Flow Rate 1 0 mL min Chart Speed 20 cm min Detector Sensitivity 0 5 1 0 AUFS Time Constant 0 2 seconds or less dilute test mixture 1 to 10 in mobile phase inject 2 to S pl of this solution Performance Monit
5. a Peak Width at Half Height 5 54 Peak Width at 4 4 Peak Height 5 Sigma 25 0 Tangent 16 0 Dr Shulamit Levin Medtechnica Plate Count Efficiency of the Separation A Plate Count Actually Is a Determination Of Both The Column AND Instruments Performance Performance Monitoring Band Spreading Band Spreading Impacts Chromatographic Performance The Greater The Band Spreading The Poorer The Performance ie Resolution Band Spreading Contains Both An Instrument AND A Column Contribution Extra Column Band Spreading The Observed Bandwidth TOT Sum of the Bandspreading Contributions Column COL Extra Column EC Instrument components O2 02 02 TOT COL EC Performance Monitoring Extra Column Band Spreading Instruments Contribution 1 Injection Volume 2 Injector 3 Connection Tubing a from Injector to Column b from Column to Detector c Endfittings and Frits 4 Detector Volume Dr Shulamit Levin Medtechnica Band Spreading Column Contribution 62 optimized by choosing the correct column COLUMN and conditions Instuments Contribution Extra Column o2 o2 02 02 02 EC TUBING CONNECTIONS INJECTORS DETECTORS Connectors fa 0 090 m a Parker _ Bis Style Other WaterSRColumns 0 130 lt a Waters _ MD Style Installation and Equilibration v Make sure column inlet connected correctly v Make sure nut and ferrule are seated A
6. 8 00 30 00 Minutes Extraneous Peaks Unusual Phenomena Extraneous Peaks Problems with Baseline Isocratic LC Extra Peak Sharp Contaminant Extraneous Peaks Extraneous Peaks Sample Blank PG 2 919 BHA 6 896 BHT 10 633 TBHQ 4 525 U Isocratic LC Broad Peak from Previous Injection or Injector Contamination Dr Shulamit Levin Medtechnica 14 Isocratic LC Negative Peak often occurs in lon Pairing Sample Solvent Degas Solvents SEANA Vacuum Time 1 minute Dr Shulam t Levin Medtechnica Installation and Equilibration V Connect Column Inlet v Purge Column at Low Flow Rate To Waste Then Connect to Detector begin flow of analytical columns at 0 1 ml min increase by 0 2 ml min increments every 30 seconds until final analytical flow rate is reached CO Ca Ol L mobile phase flow direction 5 a y Q _ Soe Guard HPLC Column HPLC Pump Manual Column Injector Detector solvent Degassing Precautions 1 Degas solvents prior to adding modifiers 2 Helium sparge is good as long as solvent doesn t change due to volatility of solvents and or additives 3 Solvents should be degassed daily 15 BASELINE TROUBLESHOOTING AA ea Noisy baseline Cyclic N AA A AA Synchronous noise Spikes NAS ANANN No peaks Asynchronous noise Drift Posit
7. Troubleshooting Common HPLC Problems http www hplc1 com shodex english dd htm A E Troubleshooting Gm Detector Control amp Data Processing Waste Fraction Collector Dr Shulamit Levin Medtechnica Performance Monitoring Use Your Test Method Known Performance Monitor at least One Peak in one injection Plate Count Peak width relative to RT Peak Asymmetry Retention Time and or Retention parameter Relative Retention Time for Critical Pair of Analytes Peak Response Inject Multiple Runs Precision at least 5 injections Accuracy Use Control Samples Troubleshooting Problem CHEMISTRY Hardware Software COLUMN GUARD COLUMN SOLVENT PUMP SAMPLE INJECTOR DETECTOR INTEGRATION Performance Monitoring Use Your Test Method Known Performance Monitor at least One Peak in one injection Plate Count Peak width relative to RT Peak Asymmetry Retention Time and or Retention parameter Relative Retention Time for Critical Pair of Analytes Peak Response Inject Multiple Runs Precision at least 5 injections Accuracy Use Control Samples Performance Monitoring Column Efficiency N the number of Theoretical Plates a is a constant depending on the Method used t retention time of peak W the peak width time units at a given peak height 2 N a t W METHOD
8. a peak WHAT TO DO Inject acetone solution to make a peak 17 N _ ee NY VY NEGATIVE amp POSITIVE PEAKS INSTRUMENTAL CHEMICAL Air bubbles passing through cell 22935 mobile pias Some eluting compounds You re using an RI detector absorb less than solvent May be normal since peak direction is a Use a different or cleaner function of solvent refractive index differential from mobile phase All peaks negative polarity wrong Reverse leads or change detector polarity All peaks negative You re using indirect UV Change polarities or reverse leads Basic assumptions The HPLC is plugged in and turned on Solvent is in the reservoir The pumps are primed and in good working order The HPLC is plumbed and wired correctly The detector has a good lamp in it The solvent bottle doesn t have a vacuum on it You re not using acetone for solvent at 195 nm You re not injecting rocks You re not doing a water to hexane gradient Your re not trying to detect sugars at 254 nm You re not mixing MEOH and water without degassing You re not sparging with nitrogen or air You re not running water through a silica column Solvent pH is not 13 on a silica base column You re not running a 1M NaCl to 100 ACN grad You re not doing gradients with an RI detector CL You re RI is not under the air conditioner vent CH No buffer stalagtites on your pump heads HCI vapors are not blowing onto y
9. e Bases Increased Retention with Increasing pH e 10 Change in Retention per 0 1 pH Units Dr Shulamit Levin Medtechnica Reversed Phase Retention Behavior of Acidic em Relative to Changes 1 pH Unit pH Control AZT Robustness Testing Ur ionjzed Acid 6 Methanol 6 THF 1 pH unit 91 un ionized g 25 o 8 20 pK ae reproducibility a Imp 1 O do 1 pH unit 91 ionize ae 8 Imp 3 fo 008 85 lonized Acid N pH 2 5 0 006 imp 2 N o 1 2 3 4 A 6 7 8 9 10 1 12 20045 Phoebe Tran ai pH N 7 N RN XJ N I K J Imp 4 Waters Corporation 2000 000 Nee TT a az DR ee E 01900 Reversed Phase Retention Behavior of Basic i j 0 002 AAA Compounds Relative to Changes in pH 2 Po Imp 1 gt 2 pH units provides stable Un ionized Base cae Imp 3 31 retention better reproducibility 0 008 Imp 2 pH 2 7 lt at flat portions of curve 0 006 n N 5 I N 5 0 004 I 5 20 0002 J A l Imp 4 154 o oo0 1 WW SA J pKa 0 002 104 Q o O 54 lonized Base 0 T T T T T T T T T T 0 il 2 3 4 5 K T 8 9 10 11 12 1 gt pH 2 gt Phoebe Tran fers Corporation 2000 Changing Retention Times Retention times getting shorter after each injection COLUMN REGENERATION Sample analytes can adhere to and cover acti
10. ive amp negative peaks ai a_i LS SYNCHRONOUS NOISE ALMOST ALWAYS CAUSED BY THE PUMP Air in pump head Prime pump and degas solvent Check valve problem Rebuild or replace Broken plunger Replace blame it on someone else Mixing problem Increase system volume Electrical noise Change circuits remove source Dr Shulamit Levin Medtechnica eh FO NOISY BASELINE INSTRUMENTAL CHEMICAL WEAK DETECTOR LAMP TRASH ELUTING OFF COLUMN Flush column with strong solvent LEAKS Stop leaks Replace fittings DETECTOR CELL DIRTY Flush with 6N nitric acid GAS IN MOBILE PHASE Degas solvent GAS BUBBLE IN DETECTOR CELL Put 009 tubing after detector not RI ELECTRONIC NOISE Remove source Shield cables Clean contacts SENSITIVITY TOO HIGH Lower sensitivity Adjust gain _ lt _ __N ASYNCHRONOUS NOISE BUBBLES Degas mobile phase GAS CAUGHT IN DETECTOR Degas mobile phase Put backpressure on cell LEAKS Fix leaks replace fittings MIXING PROBLEMS Increase system volume PLUGGED LINES Remove plug flush system ELECTRICAL PROBLEMS Remove source change circuits 16 BASELINE DRIFT INSTRUMENTAL GRADIENT SOLVENT B ABSORBS MORE THAN SOLVENT A Try anew mobile phase use baseline subtraction SOLVENT CHANGING GAS ABSORPTION EVAPORATION Helium sparge enclose solvents SOLVENT LEAKS Tighten replace fittings THERMAL EFFECTS ESPECIALLY RI CONDUCTIVITY ECD Cell temperature regulation BACKPRESSURE CHANGES
11. lumn for injection 551 on analytical column Extension of column lifetime with Guard Column using a mixture of sulfa drugs as the sample Variable Reported Concentrations Problems with Peak Response Linearity Test of Concentrations Check Injector Use Standards Multiple Injections Same Vial Syringe Problem or If Only 1st Injection Low Septa Problem Different Vials Evaporation Degradation Injection Volume Test Weight before and after injection RSD lt 5 15 Integration Software Electronic Peak Generator Poor Peak Shape 0 010 AU 0 005 Detector Cell Problem _ Le Lamp Failing 0 000 51 8 52 0 522 52 4 52 6 52 8 Dr Shulamit Levin Medtechnica Performance Monitoring Use Your Test Method Known Performance Monitor at least One Peak in one injection Plate Count Peak width relative to RT Peak Asymmetry Retention Time and or Retention parameter Relative Retention Time for Critical Pair of Analytes Peak Response Inject Multiple Runs Precision at least 5 injections Accuracy Use Control Samples Troubleshooting your UV detector Reference Energy Sample Energy Absorbance gt Offset 13 0 000 0 00 2 00 4 00 6 00 8 00 10 00 12 00 14 00 16 00 18 0020 0022 0024 0026 002
12. oring Impact of System Band Spread on a Plate Count System with 7Oul Band Spread gt gt 10 000 plates System with 130ul Band Spread gt gt 8 000 plates On the Same Column Assumption lt 40 loss in resolution at k 5 and N 10 000 and lt 20 loss in resolution at the preferred value Performance Monitoring Incorrect Sample Solvent Use Your Test Method Known Performance Sample in MeOH AU 0 003 Minocycline 0 002 Tetracycline Demeclocycline Monitor at least One Peak in one injection Plate Count Peak width relative to RT Peak Asymmetry Retention Time and or Retention parameter Relative Retention Time for Critical Pair of Analytes sa Sample in HPLC Mobile Phase Peak Response 0 005 0 1 TFA 4 ACN and 5 MeOH in Water Minutes Minocycline Tetracycline AU 0 Demeclocycline Inject Multiple Runs Precision at least 5 injections Accuracy Use Control Samples oo Fr Silica Solubility Curve 240 Column Use 220 200 180 Silica pH 2 8 140 v Instability of bonded phase at low pH 120 At pH lt 2 the functional 100 group is stripped v Elevated temperatures decrease column lifetime v C18 approximately 1000 times more stable than CN ppm solubility of Silica in water Dr Shulamit Levin Medtechnica 6 Column Collapse voided column Mass Overload encountered when mass injected onto column exceeds a certain limit
13. our HPLC You re having a wonderful time DOG eae e DB oo oo a a x a CO O 0 0 N SD Vv a WN O Dr Shulamit Levin Medtechnica Strange things can happen Radio transmitters can cause baseline noise Contaminated helium bottles and lines can cause noise System components can get coated with impurities Solvent vendors can misname solvent bottles Some filters can introduce particulates Things not to do Plug the outlet of your RI detector Flush your system with methanol after running buffer Inject samples that may precipitate in the eluent Run long durations with HCI on your stainless steel HPLC Filter organic solvents through aqueous filters Spill buffers onto HPLC electronics Try to change the column frits while it still has pressure in it Store THF on the shelf uncapped for weeks Pump cyclohexane above 2000 psi Tightly seal your mobile phase container Cut tubing with a wire cutter 18
14. ve REVERSE PHASE functional group sites making a shorter column Wash with unbuffered mobile phase Wash with 100 water Wash with methanol or ACN Wash with THF or IPA Wash with methylene chloride 4 Before injection 2 3 4 5 6 Wash with N Heptane 7 8 9 1 E 15 cm Wash with methylene chloride Wash with methanol or ACN Wash with water 0 Return to solvent After injection 12cm Covered functional groups Dr Shulamit Levin Medtechnica 10 Installation and Equilibration v Purge column with 10 column volumes of mobile phase to be used in analysis gt gt gt 4 6x150mm 25mL v Reversed Phase C18 etc columns equilibrate quicker than Normal Phase columns magnitude of ten v Normal phase columns silica or alumina may take several DAYS at flow rates of 1 0 ml min Solvent Viscosities Dimethyl sulfoxide 0 0 45 Methanol G Remember Some mixtures are more viscous than either pure solvent 50 50 MeOH H2O is almost 2x 1 Dr Shulamit Levin Medtechnica Installation and Equilibration Internal Diameter mm Length mm Column Volume mL i 47 Solvent Viscosities C DR Meines SO I CIS SO E Remember Some mixtures are more viscous than either pure solvent 50 50 MeOH H2O is almost 2x 11 Column Protection Major cause of column deterioration is contamination Use of guard columns may increase column life time to gt 10 000 analyses

Troubleshooting Common HPLC Problems

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