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Waters HPLC Diagnóstico Y Corrección De Anomalías

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©2007 Waters Corporation
Diagnóstico y corrección
Diagnóstico y corrección
de anomalías
de anomalías
HPLC /UPLC
HPLC /UPLC
HPLC /UPLC
HPLC /UPLC
Cerdanyola, , Enero
Cerdanyola
Enero 2014
2014
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Manuales relacionados para Waters HPLC

Resumen de contenidos para Waters HPLC

  • Página 1 Diagnóstico y corrección Diagnóstico y corrección de anomalías de anomalías HPLC /UPLC HPLC /UPLC HPLC /UPLC HPLC /UPLC Cerdanyola Cerdanyola, , Enero Enero 2014 2014 ©2007 Waters Corporation...
  • Página 2 Potential sources of chromatographic problems — Performance monitoring — Band spreading Section 2 — Peak shape problems Section 3 Section 3 — Retention time problems Section 4 — Miscellaneous problems Section 5 — Column protection & baseline troubleshooting ©2007 Waters Corporation...
  • Página 3 Troubleshooting Strategy Problem! Try to simplify: Inspect the chromatography Try to categorize Troubleshoot the easiest to fix items first CHEMISTRY MECHANICAL PUMP COLUMN INJECTOR GUARD COLUMN DETECTOR SOLVENTS DATA COLLECTION ADDITIVES CONNECTIONS SAMPLE & VIALS TUBING VIALS ©2007 Waters Corporation...

  • Página 4: Troubleshooting Overview

    Troubleshooting overview Troubleshooting overview •Linearity •Retention time •Area •Resolution •Solvent- Pump Autoinjector Colum Detector •reservoir •Tailing •Plate count •Noise/drift ©2007 Waters Corporation...
  • Página 5 A powerful trouble shooting tool that helps customers quickly eliminate and identify causes of failure. Eliminate own samples and mobile phase as the cause of error. Identify column or system issues based on neutrals standard performance and system information (ACQUITY console) • ©2007 Waters Corporation...
  • Página 6 Mezcla de 9 componentes que permiten verificar de forma exhaustiva sistemas LCMS o MS en un amplio rango de métodos y condiciones. Mezcla desarrollada por Waters en el centro de I&D y producción de Manchester, y utilizada para la verificación de la instrumentación del Laboratorio de demo.
  • Página 7 Los criterios y especificaciones establecidos permiten determinar si el resultado del test QCRM indica un correcto funcionamiento del sistema. Criterios típicos del QCRM para sistemas MS son: 1)Exactitud de masa 2)Sensibilidad 3)Respuesta 4)Rango del tiempo de retención o reproducibilidad 5)Area de los picos 6)Resolución de los picos ©2007 Waters Corporation...

  • Página 8: Componentes En El Qcrm Para Lcms

    Componentes en el QCRM para LCMS Componentes en el QCRM para LCMS ©2007 Waters Corporation...
  • Página 9 Criteria and specifications should allow customers to determine if the QCRM results indicate that their system and column is functioning as expected. Typical criteria might include: oRetention time range or reproducibility Peak area range or reproducibility Peak tailing range Peak resolution Plate Count Sensitivity Response ©2007 Waters Corporation...
  • Página 10 Experiment procedure and sample Experiment procedure and sample chromatogram chromatogram 0.030 0.020 0.010 0.000 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Minutes •Sample set run three times a day. ©2007 Waters Corporation...
  • Página 11 1.13 11009 Average 1.631 153239 1.12 10753 %RSD 0.44 %RSD 0.47 Plate Acenaphthene Retention Area Tailing Plate Count Acenaphthene Retention Area Tailing Count Average 2.893 101332 1.08 10436 Average 2.893 99538 1.08 10220 %RSD 0.44 %RSD 0.44 ©2007 Waters Corporation...
  • Página 12 1.12 10751 Average 1.633 155515 1.13 11009 %RSD 0.45 %RSD 0.44 Plate Plate Acenaphthene Retention Area Tailing Acenaphthene Retention Area Tailing Count Count Average 2.893 101332 1.08 10436 Average 2.893 100707 1.08 10247 %RSD 0.44 %RSD 0.44 ©2007 Waters Corporation...
  • Página 13 Neutral compounds for most analytical 186006360 HPLC/UPLC assays Reversed-Phase Suitability Neutral, Acid, Base for most 186006363 Standard analytical HPLC/UPLC assays Preparative Chromatography Neutral, Acid, Base for most UV and MS 186006703 purification HPLC assays AutoPurification Dye Mix Visible markers for fraction collection...
  • Página 14 – moderate peak width Blue analyte: Slowest – BROADEST/MOST DILUTED BAND -- Waters c 1998 Broadest Peak (longer on the column – takes more mobile phase to sweep it out of the column -- broader the peak)
  • Página 15 Where Band Spreading Occurs Where Band Spreading Occurs Simple Isocratic HPLC System Simple Isocratic HPLC System Mobile Phase HPLC Column (Stationary Phase) Data Data Injector AutoSampler Sample Pump Detector Where Band spreading occurs Waste {Flow path of sample from injector to the detector}...
  • Página 16 Where Band Spreading Occurs Where Band Spreading Occurs in gradient systems in gradient systems Mobile Phase B HPLC Column (Stationary Phase) Data Pump B Pump B Injector •Mixer AutoSampler Sample Detector Pump A Mobile Phase A Waste Where Band spreading occurs {Flow path of sample from injector to the detector} ©2007 Waters Corporation...
  • Página 17 Since there is no down column, V is close to 0 close to 0 Measure Width 4.4% (W) and Convert to Volume in l Auto Zero Problem Base line off-set ©2007 Waters Corporation...
  • Página 18 10 11 12 13 14 15 16 17 18 19 20 Retention Factor Note: You will not get optimum plate All Columns “would” count performance on standard provide 10,000 plates -- instruments as you reduce the column ID, Impact greatest for early eluters especially below 2.0mm ID ©2007 Waters Corporation...
  • Página 19 Detector flow cell design/reduced volume — Reduce injector sample loop volume — Use 0.005” ID tubing/short lengths — Perfect (pre-cut) connections (with variable depth inlet) — Time Constants <0.2 — Reduce the number of tubing connections to a minimum ©2007 Waters Corporation...
  • Página 20 Most common causes of Most common causes of extra band spreading extra band spreading Incorrect tubings — Diameters — Lenghts Incorrect fittings ©2007 Waters Corporation...
  • Página 21 System Bandspreading on System Bandspreading 0.009" I.D. W = 68 µ µ µ µ l 0.020" I.D. W = 127 µ µ µ µ l 0040" I.D. W = 400 µ µ µ µ l 1 minute ©2007 Waters Corporation...
  • Página 22 Effect of Wrong Detector cell on Effect of Wrong Detector cell on System Bandspreading System Bandspreading Prep Cell Bad RI Cell W = 236 µ µ µ µ l Analytical Cell Good RI Cell W = 87 µ µ µ µ l ©2007 Waters Corporation...
  • Página 23 7.00 7.50 8.00 7.00 7.50 8.00 • Improved plumbing • Customer’s plumbing • Replaced 10 connections between • Lack of sensitivity the injector, switching valve and MS • Removed 4 feet of extra 0.005 inch id tubing ©2007 Waters Corporation...
  • Página 24 Potential sources of chromatographic problems — Overall troubleshooting strategy — Performance monitoring Section 2 — Peak shape problems Section 3 Section 3 — Retention time problems Section 4 — Miscellaneous problems Section 5 — Column protection & baseline troubleshooting ©2007 Waters Corporation...

  • Página 25: Tabla De Contenido

    Contaminated in-line filter/guard column * — Column destroyed — Secondary interactions — Incorrect sample solvent — Temperature — — Column overload Column overload Mass overload Volume overload — Other extra-column effects Sampling rate* Detector time constant/filter value* ©2007 Waters Corporation...
  • Página 26 Tubing & fittings — Silanol activity — — Column voided Column voided — — Column overload Column overload — Contaminated in-line — Co-elution filter — Elution from previous — Injector problem injection Chemical problem — Sample solvent problem ©2007 Waters Corporation...
  • Página 27 Peak Shape Problems Peak Shape Problems double peaks fronting peaks • All Peaks Affected All Peaks Affected — COLUMN Voided — COLUMN DESTROYED (pH <2 washes off functional group) pH >8 dissolves silica base ©2007 Waters Corporation...
  • Página 28 Normal column Normal column Packing Material Flush “Well packed column” ©2007 Waters Corporation...
  • Página 29 Good Results Good Results “Well packed column” ©2007 Waters Corporation...
  • Página 30 Column collapse Column collapse Packing Material Settled “Voided column” ©2007 Waters Corporation...
  • Página 31 Column Collapse (Voiding) Column Collapse (Voiding) (shock/high pH/‘old’ (used) packing) (shock/high pH/‘old’ (used) packing) All Peaks Distorted Voids - high back pressure, distorted and/or double peaks ©2007 Waters Corporation...
  • Página 32 Limitations of Silica Based pH Limitations of Silica Based Packing Materials Packing Materials Hydrolysis of Dissolution of Bonded Ligand Silica particle Hybrid / Polymeric -- Wider pH Range ©2007 Waters Corporation...

  • Página 33: Contaminated In-Line Filter/Guard Column

    Peak Shape Problems Peak Shape Problems Contaminated in-line filter/guard column Column destroyed Secondary interactions Incorrect sample solvent Temperature Column overload Column overload — Mass overload — Volume overload Other extra-column effects — Sampling rate — Detector time constant/filter value ©2007 Waters Corporation...

  • Página 34: Secondary Interactions

    Poor Peak Shape for Basic Compounds due to Poor Peak Shape for Basic Compounds due to Secondary Interactions Secondary Interactions Integration errors Acids and Neutrals have Reduced resolution Good peak shape -- Reduced sensitivity Basic Analytes “Tail” Minutes ©2007 Waters Corporation...
  • Página 35 Tailing of Bases Tailing of Bases -- -- Chemical Problem Chemical Problem (Column Brand/pH (Column Brand/pH Different Silanol Activities for Different Column Brands Neutral Conventional C Conventional C Base Base Neutral Current Generation C Time (min) ©2007 Waters Corporation...
  • Página 36 Phase pH < 3 O- Si O- Si O-Si O-Si Si - OH O- Si O-Si O- Si O-Si O- Si O-Si Mobile O- Si Phase pH > 3 O--Si O- Si O-Si Si – O Base Base ©2007 Waters Corporation...
  • Página 37 Column comparison Column comparison Waters Bondapak™ C18 1973, USP TF = 10 Acenaphthene Amitriptyline Minutes Waters Xbridge RP18 2008, USP TF = 1.1 10 15 Minutes ©2007 Waters Corporation...

  • Página 38: Incorrect Sample Solvent

    Peak Shape Problems Peak Shape Problems Contaminated in-line filter/guard column Column destroyed Secondary interactions Incorrect sample solvent Temperature Column overload Column overload — Mass overload — Volume overload Other extra-column effects — Sampling rate — Detector time constant/filter value ©2007 Waters Corporation...
  • Página 39 Effect of sample solvent Effect of sample solvent Sample in methanol Minocycline Demeclocycline Tetracycline Minutes Sample in mobile phase Minocycline (0.1% TFA, 4% ACN, 5% MeOH) Tetracycline Demeclocycline Minutes ©2007 Waters Corporation...
  • Página 40 Peak Shape Problems Peak Shape Problems Contaminated in-line filter/guard column Column destroyed Secondary interactions Incorrect sample solvent Temperature Column overload Column overload — Mass overload — Volume overload Other extra-column effects — Sampling rate — Detector time constant/filter value ©2007 Waters Corporation...
  • Página 41 Column temperature equilibration Column temperature equilibration 70°C 70°C 70°C 70°C 22°C 45°C 65°C 70°C 70°C ©2007 Waters Corporation...
  • Página 42 Peak Shape Problems Peak Shape Problems Contaminated in-line filter/guard column Column destroyed Secondary interactions Incorrect sample solvent Temperature Column overload Column overload — Mass overload — Volume overload Other extra-column effects — Sampling rate — Detector time constant/filter value ©2007 Waters Corporation...
  • Página 43 Column/Volume Overload Column/Volume Overload 10 L 100 L 300 L 500 L Effect of injection volume on peak distortion ©2007 Waters Corporation...
  • Página 44 Volume Overload Volume Overload Note : Peak starts at same time ©2007 Waters Corporation...
  • Página 45 (4.6 X 50mm ODS) Wider peaks first Wider peaks first observed at low Minutes retention Peak position shifts 6.25 g injected in 500 L to higher retention % of column volume: 62.5 in proportion to the injection volume Minutes ©2007 Waters Corporation...
  • Página 46 Minutes Preparative load of 25 mg 25 mg injected generates mass overload peak shape Minutes Note that the back of the peaks of the analytical and prep loads are at the same retention (-------) ©2007 Waters Corporation...

  • Página 47: Other Extra-Column Effects

    Peak Shape Problems Peak Shape Problems Contaminated in-line filter/guard column Column destroyed Secondary interactions Incorrect sample solvent Temperature Column overload Column overload — Mass overload — Volume overload Other extra-column effects — Sampling rate — Detector time constant/filter value ©2007 Waters Corporation...
  • Página 48 Summary Summary Peak shape problems — All peaks affected Mechanical problem Exception : Incorrect sample solvent — — Only one or two peaks affected Only one or two peaks affected Chemical problem Minutes ©2007 Waters Corporation...
  • Página 49 Potential sources of chromatographic problems — Overall troubleshooting strategy — Performance monitoring Section 2 — Peak shape problems Section 3 Section 3 — Retention time problems Section 4 — Miscellaneous problems Section 5 — Column protection & baseline troubleshooting ©2007 Waters Corporation...
  • Página 50 Content Content Retention Time problems — Reproducibility versus drifting or incorrect retention times Temperature Organic % Chemistry problem Chemistry problem Ion-pairing De-wetting (Hydrophobic Collapse) ©2007 Waters Corporation...
  • Página 51 Retention Time Problems Reproducibility, incorrect & Drifting Retention - Equilibration - Solvent Composition - Stationary Phase - Temperature Stability - pH Control - pH Control - Column Contamination - Ion Pairing - De-wetting/Hydrophobic Collapse (Low % Organic) ©2007 Waters Corporation...
  • Página 52 — C8 – less retention — C18 – more retention Temperature Problem — warmer – less retention, — colder – more retention % Organic In Mobile Phase — more – less retention, — less – more retention) ©2007 Waters Corporation...
  • Página 53 Content Content Retention Time problems — Reproducibility versus drifting or incorrect retention times Temperature Organic % Ion-pairing De-wetting (Hydrophobic Collapse) ©2007 Waters Corporation...
  • Página 54 Effect of Temperature Effect of Temperature Reversed phase chromatography Reversed phase chromatography Reduction of Retention with Increasing Temperature 1% to 2% Change per 1° Celsius Shifts in Selectivity Usually Small Shifts in Selectivity Usually Small ©2007 Waters Corporation...
  • Página 55 Effect of Temperature (Isocratic Effect of Temperature (Isocratic Separations) Separations) •Efficiency •Back-Pressure •1160 psi •N=2250 •60 •Higher Temperature: •Higher Temperature: •50 •Shorter Run Time •Sharper Peaks •Better Sensitivity •40 •Lower Back Pressure •30 •1920 psi •N= 1680 •1.00•2.00•3.00•4.00•5.00•6.00•7.00•8.00•9.00•10.00 •Minutes ©2007 Waters Corporation...
  • Página 56 Effect of Temperature (Isocratic Separations (Isocratic Separations 23.5°C •Higher Temperature: Temperature: •Shorter Run Time •5 •10 •15 •20 •25 •30 •Sharper Peaks •Minutes 26.5°C •Better Sensitivity •Note selectivity change •Lower Back Pressure •5 •10 •15 •20 •25 •30 •Minutes ©2007 Waters Corporation...
  • Página 57 •3.5 m, 4.6 mm x 50 mm •Mobile phase: •0.10 •25% MeOH, 65% Water, •4 •10% Ammonium •50°C •5 •0.05 •bicarbonate buffer, pH 9 •0.00 •0.00 •1.00 •2.00 •3.00 •4.00 •5.00 •6.00 •7.00 •8.00 •9.00 •10.00•11.00•12.00 •Minutes ©2007 Waters Corporation...
  • Página 58 Content Content Retention Time problems — Reproducibility versus drifting or incorrect retention times Temperature Organic % Ion-pairing De-wetting (Hydrophobic Collapse) ©2007 Waters Corporation...
  • Página 59 Retention Time Change 30.00 30.00 of 5% to 15% per 1% of 5% to 15% per 1% Change in Solvent 25.00 Composition 20.00 15.00 Bonded Phase Collapse in 10.00 High Water Content 5.00 (discussed later) 0.00 % Methanol ©2007 Waters Corporation...
  • Página 60 Change in Solvent Composition reversed phase chromatography reversed phase chromatography •Can be caused by organic component evaporating from reservoir •More organic •shorter retention •1 •2 •3 •4 •Minutes •Less organic •Longer retention •1 •2 •3 •4 •Minutes ©2007 Waters Corporation...
  • Página 61 Questions???? Questions???? •Mobile •How difficult is it to • Phase make up a liter of • Preparation •60/40 MeOH/Water •Mobile Phase???? •How would You prepare this •mobile phase???? •Would we all get the same result???? ©2007 Waters Corporation...
  • Página 62 •Options A and A’: Adding organic/aqueous directly to measuring vessel (not recommended) • Need to specify which solvent is added first: e.g. H2O to MeOH or MeOH to H • • •Standardize pH Adjustment – ie; BEFORE organic is added ©2007 Waters Corporation...
  • Página 63 •Options A and A’: Adding organic/aqueous directly to measuring vessel (not recommended) • Need to specify which solvent is added first: e.g. MeOH to H O or H O to MeOH •Options B: Separate volumetric measurement • • ©2007 Waters Corporation...
  • Página 64 Need to specify which solvent is added first: e.g. MeOH to H O or H O to MeOH weighing is most accurate •Options B/C: Separate volumetric measurement or • • •Standardize pH Adjustment – ie; BEFORE organic is added ©2007 Waters Corporation...
  • Página 65 Need to specify which solvent is added first: e.g. MeOH to H O or H O to MeOH •Options B/C: Separate volumetric measurement or weighing is most accurate • • •Standardize pH Adjustment – ie; BEFORE or AFTER organic is added ©2007 Waters Corporation...
  • Página 66 Content Content Retention Time problems — Reproducibility versus drifting or incorrect retention times Temperature Organic % Ion-pairing De-wetting (Hydrophobic Collapse) ©2007 Waters Corporation...
  • Página 67 Acids: Acids: Reduced Retention with Increasing pH Reduced Retention with Increasing pH Bases: Increased Retention with Increasing pH Up to 10% Change in Retention per 0.1 pH Unit (Largest shift within +/- 1 pH unit of pKa) ©2007 Waters Corporation...
  • Página 68 Relative to Changes in pH Relative to Changes in pH •> ± 2 pH units provides stable retention (better reproducibility at flat portions of curve) •Non-ionized Base •Range where •pH control •is critical •pK •Ionized Base •pH •± ©2007 Waters Corporation...
  • Página 69 •0.1 •pH •0 •2 •4 •6 •8 •10 •12 •14 •(Ionized) •N •B2 •pH 5.5 •B1 •N •A, •B2 •A •pH6.0 •pH: Powerful tool in methods development for Selectivity – Range where pH control will be critical ©2007 Waters Corporation...
  • Página 70 Test for Robustness (+/ Test for Robustness (+/- - 0.2 pH Units) 0.2 pH Units) •AZT Robustness testing •Imp1 •Imp3 •pH 2.3 •Imp2 •Imp4 •Imp1 •Imp3 •Imp3 •Imp2 •pH 2.5 •Imp4 •Imp1 •Imp3 •Imp2 •pH 2.7 •Imp4 ©2007 Waters Corporation...
  • Página 71 Content Content Retention Time problems — Reproducibility versus drifting or incorrect retention times Temperature Organic % Ion-pairing De-wetting (Hydrophobic Collapse) ©2007 Waters Corporation...
  • Página 72 Pairing Agent at Low Concentration Nearly Independent of the Concentration of the – Pairing Agent at High (~10mM/L) Concentration Long Equilibration Times Due to Adsorption of the Reagent on the Stationary – Phase (can be up to 500 Column Volumes) ©2007 Waters Corporation...
  • Página 73 Content Content Retention Time problems — Reproducibility versus drifting or incorrect retention times Temperature Organic % Chemistry problem Chemistry problem Ion-pairing De-wetting (Hydrophobic Collapse) ©2007 Waters Corporation...
  • Página 74 •Analyte •retention of the analyte •to take place. •If the pores are dry, the analyte cannot get into the pores and it will not be retained by the chromatographic surface. What influence does this have on chromatography? ©2007 Waters Corporation...
  • Página 75 “Hydrophobic Collapse •Mobile phase: Aqueous 0.1% Acetic Acid •Initial •Amoxicillin Column pre-wetted Column pre-wetted with organic •1500 psi •After Flow Stoppage (Pore de-wetting 100%) •1500 psi •0 •2 •4 •6 •8 •10 •Minutes •Vo: No retentivity for analyte ©2007 Waters Corporation...

  • Página 76: De-Wetted Pore

    What is De What is De- - wetting or “Hydrophobic wetting or “Hydrophobic Collapse?” Collapse?” •Low % organic or pure aqueous mobile phase •C18 Silica- Gel Particle Pores •Wetted Pore •De-wetted Pore ©2007 Waters Corporation...
  • Página 77 How does flow stoppage cause this problem How does flow stoppage cause this problem with an HPLC column? with an HPLC column? •Analytes •Analytes •Stopped flow with no pressure on the •At flow with pressure on the mobile phase, pores de-wet. Restart flow, mobile phase.
  • Página 78 Chains folding or collapsing back Chains folding or collapsing back down to the particle surface, thereby not being fully extended to interact, or retain the analyte. •In reality, it is the de-wetting of the pores which causes the loss of retentivity. ©2007 Waters Corporation...
  • Página 79 Difficult to use pressure to force aqueous mobile Difficult to use pressure to force aqueous mobile phase back into pores Typically not practical because column outlet is at atmospheric pressure •Inlet •Outlet •2200 psi •P •1 atm (14.7 psi) •Length ©2007 Waters Corporation...
  • Página 80 Reversed Reversed- - Phase Column Selection Phase Column Selection Considerations Relating to the Risk of De Considerations Relating to the Risk of De- - Wetting/Hydrophobic Collapse Wetting/Hydrophobic Collapse Pore Size Ligand Density Ligand Type ©2007 Waters Corporation...
  • Página 81 Chapter 4 Chapter 4 Miscellaneous problems Miscellaneous problems Methods Development Suggestions Extraneous Peaks Mobile phase degassing Gradients Samples and Vials Samples and Vials ©2007 Waters Corporation...
  • Página 82 Changes in Retention/Resolution Changes in Retention/Resolution •Common Complaints: • “My column is not reproducible” • – “a new column gives different results” • “My chromatography changes” • “Quality Control Laboratory can’t reproduce • my results” WHY? • ©2007 Waters Corporation...
  • Página 83 •Changes in Retention/Resolution Method Development •Please remember: •HPLC Columns contain packing materials which can be which can be chemically altered chemically altered depending depending on how they are used and treated (mobile phase, pH, ion-pairing reagents, surfactants, age). •“Column History”...
  • Página 84 •(in a drawer, on the instrument, donated by a colleague) •“Regeneration” may not always bring you back to the original chromatographic surface, especially, if ligand hydrolysis (loss of ligand) or surface modification by ion-pairing reagents has occurred ©2007 Waters Corporation...
  • Página 85 •Column Product is OK but •Column Product is OK but not manufactured •use/conditions are changing reproducibly •the performance •How do you determine, and control the method development outcome? •A suggested approach which controls the results, without confounding the reasons. ©2007 Waters Corporation...
  • Página 86 A BATCH* may be used to make many different LOT #’s, and many thousands of columns Approve and Ship • •* Note: Some column manufacturers buy packings and only pack columns •They may have little if any control over chromatographic performance. ©2007 Waters Corporation...
  • Página 87 (C & D) Select column A of the “2 from the SAME BATCH” of new columns and develop method conditions •* Especially important for validated methods which • will be used for many years or in different locations ©2007 Waters Corporation...
  • Página 88 NOT THE SAME – then you “modified” column A while you were developing the method. This necessitates starting again. Even a new column from the same batch will not duplicate your chromatography. • Move back to step 2 ©2007 Waters Corporation...
  • Página 89 • If the results are DIFFERENT – the manufacturer does NOT have adequate batch to batch reproducibility. You will need to work with that column vendor to get more reproducible columns or choose a new column vendor/manufacturer. ©2007 Waters Corporation...
  • Página 90 Robustness •This approach provides you with an • early assessment of method robustness during the initial stages of separation during the initial stages of separation development whilst allowing you to identify reproducibility problems that may be occurring. ©2007 Waters Corporation...
  • Página 91 (especially if the ligand was hydrolyzed off the old column) old column) The new column will have more ligand, and therefore changed retention characteristics. •During Methods Development, running a “used” column •can sometimes show degree of robustness ©2007 Waters Corporation...
  • Página 92 •Tips from Case Studies Always use “New Columns” for initial method development work method development work •Avoid having to redo the development work all over again ©2007 Waters Corporation...
  • Página 93 (pH control and % organic are especially important for some methods) Differences will often be observed for a mobile phase prepared in a single reservoir vs. “same” mobile phase made by blending lines using a proportioning/mixing valve ©2007 Waters Corporation...
  • Página 94 Content Content Methods Development Suggestions Extraneous Peaks Mobile phase degassing Gradients Samples and Vials Samples and Vials ©2007 Waters Corporation...
  • Página 95 Extraneous Peaks Extraneous Peaks Isocratic LC - Extra Peak – Sharp - Contaminant •Contaminant(s) from; sample, vial, septum, injector etc… •Isocratic Method: extra peak with the same peak shape and width , •the contaminant came from this injection ©2007 Waters Corporation...
  • Página 96 Extraneous Peaks Extraneous Peaks Isocratic LC - Broad -Peak from Previous Injection ©2007 Waters Corporation...
  • Página 97 Prevent airborne contaminants from entering the solvent Clean laboratory glassware properly — First, rinse it with organic solvent and then water. — Next, rinse it with the solvent that will be put into it. — DO NOT WASH GLASS BOTTLES IN DETERGENT ©2007 Waters Corporation...
  • Página 98 — Be aware that tubing made of polymers (such as polyvinylchloride, Be aware that tubing made of polymers (such as polyvinylchloride, or PVC) may contain plasticizers or other contaminants. Wear particulate-free, powder-free, non-latex gloves Use clean columns ©2007 Waters Corporation...
  • Página 99 Contaminated mobile phase Contaminated mobile phase •4 different buffer as A solvent •(b) •(a) •HPLC grade water as A solvent •Blank gradients 5-80% ACN after •Blank gradients 5-80% ACN after 10min (a) and 30min (b) equilibration 30min (a) and 30min (b) equilibration w.
  • Página 100 Contamination sources Contamination sources ©2007 Waters Corporation...
  • Página 101 Content Content Methods Development Suggestions Extraneous Peaks Gradients Samples and Vials ©2007 Waters Corporation...
  • Página 102 Gradient Transfer Gradient Transfer Common Problem — Gradient method works excellently on the R&D HPLC System However! — The results cannot be duplicated in the Production Quality Control Laboratories Control Laboratories (resolution, retention times) Is the Column at fault? ©2007 Waters Corporation...
  • Página 103 Effect of Gradient Volume Effect of Gradient Volume •PTH Amino Acid Standards 717 Autosampler •2 mL Sample Loop F L NL •200 L Sample Loop ©2007 Waters Corporation...
  • Página 104 — High pressure or low pressure mixing/blending Even if the model and brand are the same, the EXACT SAME PLUMBING CONFIGURATION must be used for both The problems commonly arise from DIFFERENCES IN SYSTEM DELAY/DWELL VOLUMES ©2007 Waters Corporation...
  • Página 105 Check Gradient Delay/Dwell Volume of the different instruments/systems Must specify the plumbing - even ID and length of connecting tubing - should delay length of connecting tubing - should delay volumes be different, retention times etc. will also be different ©2007 Waters Corporation...
  • Página 106 Gradient Method large delay volume •1 mL/min •1.00 small delay volume •0.80 •0.60 •0.60 •AU •0.40 •Vo •0.20 •0.00 •2 •4 •6 •8 •10 •12 •14 •16 •18 •20 Minutes Change in retention time Change in resolution ©2007 Waters Corporation...
  • Página 107 Content Content Methods Development Suggestions Extraneous Peaks Gradients Samples and Vials ©2007 Waters Corporation...
  • Página 108 •Inspect the chromatography •Try to categorize •Troubleshoot the easiest to fix items first CHEMISTRY MECHANICAL VIALS SAMPLES & VIALS Dimensional Problems Contamination Jams, Pick-up Sample Stability Needle Damage Poor Injection Reproducibility Septum Dislodging Sample Evaporation LVI Assembly ©2007 Waters Corporation...
  • Página 109 Troubleshooting Concerns Regarding Vials Regarding Vials Vials Can Present both Chemical and Mechanical Problems Most important: What HPLC system is using the vial. Sometimes the autosampler appears to be at fault, but in reality it’s the vial. — Differences in the designs of HPLC systems determine the...
  • Página 110 •Sample Vials Vials are the least expensive component of an HPLC system, but can be the biggest contributor to problems that you encounter, such as: •Chemical Mechanical Sample draw volume reproducibility Evaporative loss Septum dislodging/Coring Spurious peaks/extractables Injector damage/Robotics Sample/analyte degradation...
  • Página 111 More alkaline than Class A available Amber/Clear glass — All amber glassware is “Type 1, Class B” — Used for light labile samples Polypropylene — A non-ionic, non-reactive plastic. Used where glass is inappropriate. Max. temperature : 135°C ©2007 Waters Corporation...
  • Página 112 •Can jam the autosampler Don’t immediately blame the needle or needle alignment Switch to pre-slit septum Less force required to pierce Choose bonded septum (LectraBond™) Septum is bonded to the cap. Eliminates dislodging Electro welded: No chemicals or adhesives ©2007 Waters Corporation...
  • Página 113 Choosing the Correct SEPTA Choosing the Correct SEPTA Wrong choice of Septum may result in : —Evaporative loss of sample —Lack of reproducibility for repetitive injections injections —Septum coring —Needle damage —Septum dislodging —Contamination Di-octylphthalate (silicone) Siloxane (silicone) ©2007 Waters Corporation...
  • Página 114 Remove cap and septum* from the vial.Perform multiple injections. Measure peak area to determine if the septum/cap is the cause. •* Some auto samplers may have to have the vial sensor disabled •Vial A •Vial A •Inj. 1 •Inj.2 •Area = 200,000 •Area = 250,000 ©2007 Waters Corporation...
  • Página 115 Switch to pre-slit PTFE/silicone septum Pre-slit septum will eliminate coring and deliver good resealing capability “Alternatively” Switch to PTFE septum •Silicone Septum PTFE will eliminate coring. However, it will not reseal •Material Lodges in Bottom Draw Needle ©2007 Waters Corporation...
  • Página 116 —Vial —Cap —Septum —Septum Most common contributor Chemical compatibility • Absorbs contaminants from atmosphere (silicone) • Observance of problem can be detection-method • dependent •Packaging and storage can be very important ©2007 Waters Corporation...
  • Página 117 Vials are manufactured from different grades of glass. Choose a vial manufactured from a different grade / class of glass Packaging used for shipping the vials can also contribute to this problem. Some autosamplers may require you to disable the optical vial cap sensor ©2007 Waters Corporation...
  • Página 118 Included in every package Included in every package - - COA ©2007 Waters Corporation...
  • Página 119 Chapter 5 Chapter 5 Column protection, baseline Column protection, baseline troubleshooting troubleshooting ©2007 Waters Corporation...
  • Página 120 Chapter 5 Chapter 5 Column protection, baseline Column protection, baseline troubleshooting troubleshooting Column use, storage & maintenance Column protection & regeneration Column protection & regeneration Baseline troubleshooting ©2007 Waters Corporation...
  • Página 121 : If our are using a buffer make sure it does not precipitate in the column when initially flushing the column w. mobile phase — Most RP column comes in 100% organic usually acetonitrile — Initially flush with water/organic and the switch to the mobile phase ©2007 Waters Corporation...
  • Página 122 Normal phase columns (silica or alumina) may Normal phase columns (silica or alumina) may take several hours at flow rates of 1.0 ml/min — Use dried solvents, or water saturated solvents or solvent with traces of propanol ©2007 Waters Corporation...
  • Página 123 (eg. MeOH or ACN) Using non-aqueous solvents minimizes hydrolysis. Some bonded phases (CN) become unstable in polar organic mobile phases. Storage in water or buffer is then ok. — Worst mobile phase for CN column is CH3CN ©2007 Waters Corporation...
  • Página 124 Columns which may be stored in water or buffered solvents: — CN columns — Ion exchangers — Aqueous SEC packings — — However: However: Prevent microbial growth by using 0.05% sodium azide in mobile phase Small quantity of organic solvent (acetonitrile 5% or methanol 10%) ©2007 Waters Corporation...
  • Página 125 Column Storage Column Storage Columns which should be stored in Mobile Phase: — Normal Phase — Organic SEC (GPC) ©2007 Waters Corporation...
  • Página 126 Filtering of the Sample — Protects against particles Filtering of the mobile phase (0,2µm filter) — Protect against particles and microbial growth Sample Cleanup through Solid Phase Extraction (SPE) — Protects against chemical interferences Limit High Back Pressures/Pulses (solvent viscosity) ©2007 Waters Corporation...
  • Página 127 Washing them off with more aggressive solvents can return performance If surface has been chemically altered, ie hydrolysis of ligands, then performance may not be restored ©2007 Waters Corporation...
  • Página 128 Cleaning efficiency is increased at 35-45ºC Reversed phase Normal phase columns: columns: 50/50 Methanol* Methanol/chloroform Ethyl acetate Hexane Mobile phase Methanol Mobile phase* • • •* Make sure mobile phase is miscible w. methanol and no salt precipitation occurs • ©2007 Waters Corporation...
  • Página 129 Column Lifetime Column Lifetime HPLC columns have a finite lifetime. Most common cause of column failure is due to high pressure, typically caused by particulates. Sources of particulate contamination — Debris from instrument seals — Particles in the sample —...
  • Página 130 Inject 100 µl DMSO while flushing the column with 100% acetonitrile Run a 10-100% B gradient where A= 0.1% TFA in water A= 0.1% TFA in water B= 0.1% TFA in acetonitrile Flush with 7M guanidine HCl or 7M urea ©2007 Waters Corporation...
  • Página 131 Content Content ©2007 Waters Corporation...
  • Página 132 Si vemos curvaturas de la línea de base, posiblemente la celda esté sucia. Limpiar la celda Equipo contaminado Si quitando la columna y con nuevas fases móviles, solo bombeando fase móvil sigue habiendo deriva limpieza del equipo (ver protocolo Mantenimiento y Limpieza celdas de detectores Acquity) ©2007 Waters Corporation...

  • Página 133: Linea De Base

    Línea plana, no picos Inexistencia de restrictor de presión a la salida Colocar restrictor de presión a la salida del del último detector detector (tubo azul). Si detras hay un detector de masas no es necesario ©2007 Waters Corporation...
  • Página 134 Vigilar conexiones a circuitos eléctricos próximos. Saturación del detector Comprobar si diluyendo la muestra sucede lo mismo. Comprobar los parámetros de detector programados en el método ©2007 Waters Corporation...
  • Página 135 Fase móvil tiene más lectura que analito Cambiar las composiciones de fase móvil No se ha inyectado Revisar suficiente volumen de muestra en vial, vial en correcta posición, test de pesada. Hacer prime de sample syringe y realizar el “sample srynge leak test” ©2007 Waters Corporation...

  • Página 136: Acquity Uplc™ Troubleshooting Tiempos De Retención

    ACQUITY UPLC™ Troubleshooting ACQUITY UPLC™ Troubleshooting Tiempos de retención Tiempos de retención ©2007 Waters Corporation...

  • Página 137: Tiempos De Retención

    -ver “ruido de base cíclico” Contaminación de columna -Pasar orgánico a la columna (tener en cuenta posibles precipitaciones!!) -limpiar o reemplazar columna -volver a inyectar -si el tr es errático: mirar solventes, contaminación de f.m o de precolumna o filtro inline ©2007 Waters Corporation...
  • Página 138 Filtro de solventes o inlets de las diferentes líneas Chequear la presión que se genera en cada una de bloqueados las líneas y cambiar tubos si es necesario Limpiar o cambiar los filtros de botella y el filtro inline del equipo. ©2007 Waters Corporation...
  • Página 139 Contaminación de columna -Limpiar columna según su documento de “care and use”. -en caso necesario reemplazar Degradación parcial de la columna, pérdida de funcionalización Realizar test de columna o pinchar un system suitability. Cambiar por nueva en caso necesario ©2007 Waters Corporation...
  • Página 140 Cambio de tubos por otros de diferente de gradiente correspondiente volumen (diámetro interno o longitud) Chequear o cambiar weak wash por una Partial loop con presión asistida con el weak adecuada y caracterizarlo solvent diferente de fase móvil ©2007 Waters Corporation...

  • Página 141: Resultados Cualitativos Incorrectos

    Resultados cualitativos incorrectos Resultados cualitativos incorrectos ©2007 Waters Corporation...
  • Página 142 Picos desdoblados o con hombros-colas Ver sección de forma de pico Weak wash contaminado Hacer una inyección a full loop, si la cromatografia es ok, el weak wash está contaminado. ©2007 Waters Corporation...
  • Página 143 No se realiza bien el gradiente. La bomba con fase Medir el caudal de cada una de las bombas por orgánica da menos caudal. Los último picos no separado. Si no escorrecto proceder a su tienen tiempo de salir. revisión. ©2007 Waters Corporation...

  • Página 144: Forma De Pico Y Consecuencias

    Needle wash solvent con demasiada proporción de Needle wash solvent debe tener composición orgánico similar a la de la fase móvil inicial Una parte del método o todo él es isocrático Probar a utilizar un métdo de gradiente ©2007 Waters Corporation...
  • Página 145 Weak wash es incompatible con la cromatografía Needle wash solvent debe tener composición y con el diluyente de muestra similar a la de la fase móvil inicial y ser compatible con diluyente de muestra. ©2007 Waters Corporation...
  • Página 146 Chequear y cambiar si es necesario las incorrectamente. Tubos mal cortados conexiones. No es necesario cortar los tubos. Se suministran cortados No está instalado el estabilizador de Instalar el estabilizador temperatura Dos compuestos o isomeros que coeluyen Modificación del método ©2007 Waters Corporation...
  • Página 147 La fase móvil absorbe más que el analito a la Escoger una fase móvil más transparente o longitud de onda elegida cambiar la longitud de onda de trabajo Problemas de frente de solvente Hacer más compatible el solvente de inyección y la fase móvil ©2007 Waters Corporation...

  • Página 148: Sensibilidad

    Chequar su solubilidad y si hace falta elevar la tiempo. temperatura del compartimento de muestras (a temperaturas bajas puede ser insoluble) Se evapora o degrada Disminuir la temperatura del compartimento de muestras Fase Móvil Fase móvil errónea (pH incorrecto, contaminada..) Preparar fase móvil nueva ©2007 Waters Corporation...
  • Página 149 Velocidad de aspiración de la jeringa demasiado alta. Cambiar la velocidad de aspiración en el método Puede coger burbujas Platos Configuración de platos errónea Comprobar configuración de los platos Temperatura Temperatura de columna demasiado baja o estabilizador Modificar la temperatura. Instalar estabilizador omitido ©2007 Waters Corporation...
  • Página 150 Poner volumen de muestra superior al volumen Vial residual Burbuja de aire en el fondo del vial, habitual en los insertos Vigilar volúmenes residuales de cada tipo de vial Usar preslit, el no usarlo puede provocar vacío al inyectar ©2007 Waters Corporation...

  • Página 151: Sobrepresión

    Desconectar conector entrada de columna Si presión baja, el problema está en columna, lavarla, cambiar frits, o renovar columna Si no baja, es anterior Desconectar del prefiltro online o precolumna Si presión baja, sustituir prefiltro o precolumna por una nueva ©2007 Waters Corporation...
  • Página 152 Desconectar filtro on line Si baja, el problema está en el filtro. Limpiar o poner uno nuevo Column manager Para saber si está en alguno de los tubos del column manager, probar con diferentes posiciones de columna ©2007 Waters Corporation...

  • Página 153: Limpieza De La Celda

    Si ya tengo la celda sucia u obturada como la puedo limpiar — Pasando agua si la suciedad se ha originado trabajando con tampones — Limpiar con soluciones ácidas. Existe un protocolo de limpieza disponible. Esta limpieza se ha de realizar sin columna. ©2007 Waters Corporation...

  • Página 154: Limpieza De Columna

    Para fases móviles con pH muy básicos, contactar con el departamento de columnas, para elaborar protocolos de lavado individualizados Para largos períodos inactivos: guardar en 20% agua, 80% orgánico y bien tapada. Nunca colgada del equipo. ©2007 Waters Corporation...

  • Página 155: Prevención De Problemas De Presión

    — Usar sales de alta calidad para preparar los tampones — Usar solventes orgánicos de grado HPLC gradiente o superior — — Limpiar con agua después de haber trabajado con tampones Limpiar con agua después de haber trabajado con tampones —...

  • Página 156: Compatibilidad De Solventes

    10mM phosphate buffer 10mM ammonium bicarbonate 50mM ammonium hydroxide 50mM ammonium acetate 0.1% Ethylenediaminetetraacetic acid (EDTA) Cleaners Phosphoric acid (=30%) Sodium hydroxide (=1M) ACQUITY UPLC™ System non-recommended Solvents Methylene-Chloride Chloroform Ethyl Acetate Toluene Chlorinated solvents: (Trichlorobenzene) Strong acids >5% ©2007 Waters Corporation...

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