Home

Successful HPLC Operation

1. Solvent PEEK Polyethylene Polypropylene PVDF Teflon Tefzel Acetaldehyde Acetic Acid 2096 1 1 1 1 1 1 Acetic Acid 8096 1 1 2 Acetic Acid glacial 1 1 1 1 1 Acetone 2 1 3 1 1 Acetonitrile 1 1 1 Ammonia 1096 1 2 1 1 1 Ammonia liquid 2 Ammonium Hydroxide 1 1 1 1 Aromatic Hydrocarbons 2 3 Benzene 2 1 Butanol 1 1 1 Chloroform 2 2 1 Cyclohexane 2 3 1 Cyclohexanone 3 3 3 1 Diethylamine 3 1 3 1 Diethylether Dioxane 1 1 Ethanol 1 2 1 1 1 Ethylacetate 1 2 1 3 1 1 Hexane Heptane 2 2 1 1 Hydrochloric Acid 10096 2 1 1 Hydrochloric Acid 2096 1 2 1 1 Isopropanol 1 1 Isopropyl Ether 1 2 3 Ketones general 1 2 2 2 Methanol 1 1 1 1 Methyl Dichloride 3 3 Nitric Acid 100 3 2 3 3 1 Nitric Acid 20 2 1 1 Perchloric Acid 2 2 1 Phosphoric Acid 100 2 2 1 Phosphoric Acid 20 1 2 1 Sodium Hydroxide 80 2 3 Sodium Hydroxide 20 1 1 1 1 Sulphuric Acid 100 3 2 2 1 1 Sulphuric Acid 40 1 1 1 1 1 THF 1 2 2 2 1 1 Toluene 2 2 1 1 Triethylamine 3 1 1 1 Compatible no adverse 2 Application dependant 3 Not compatible effect recommended No figure denotes compatibility not calculated 55 1 Compatible no adverse effect 2 Application dependant 3 Not compatible recommended No figure denotes compatibility not calculated Solvent Compatibility with PEEK Tubing at Elevated Temperature Solvent Acetic Acid Ammonia Liquid Hydrogen Sulphide gas Methane
2. Acetonitrile Benzene n Butanor i Immiscible EE Those squares shaded as immiscible Chloroform refer to solvent mixes where in some Cyclohexane H proportions two phases will be produced 1 2 Dichloroethane Dichloromethane Dimethylformamide Dimethyl Sulphoxide Dioxane Ethanol Ethyl Acetate Diethyl Ether Heptane Hexane Methanol EN Methyl t Butyl Ether Methyl Ethyl Ketone Pentane m te L n Propanol Iso Propanol Di iso Propyl Ether Tetrahydrofuran Toluene Trichloroethylene Water NENNEN il Xylene 2 o o 22 s 2 i S S oziz sis 3 o 2 2 Z amp I Sle 5 Slasial s z dc 2 28 o9 Zz g E e s 5 gt z o s f o 2 99 2 z o B2 Sr e ooo r ts gt T 2a S eias Essais sz tma iz ellis e2 o oZ e SS9gz x t eii g S 2 5 e 9 Z ezse S 82 9 Te l se 9 gr oes ELS z eliS lg sias ez cs E zizc oexttz5 gsgzzstzszzisTirzmszs 2 228 529 z oun c5o ocszrizmsissszimseoicisessusoiz e v 60 0 29 20z Xx cv t zz zr e S55 o 2 9 9 8 S Sz glas E Es sz ssssssssutoeoilt zs3s83 2 V 2 E E 2 s e 2 n ak mnm mjioloo aanasna uunaurrrzz szzizia z5u9sy x 63 Appendix C Example Routine Maintenance Log Sheet Number Issued By Detec
3. acyclic 210 230 21 000 C C 260 35 000 C C C C 219 6 500 C C C N 220 23 000 C C C 0 210 250 10 000 20 000 C C NO 300 350 Weak Nitrile C N 160 ONO 220 230 1000 2000 300 400 10 Nitro N0 210 Strong Nitroso N 0 302 100 Oxime NOH 190 5000 Pyridine E 74 80 000 95 6 000 N we 251 100 Sulfone S0 80 Sulfoxide 28 0 210 1500 Thioether S 194 4600 215 1600 Thiol SH 95 1400 51 5 6 Column Cleaning and Regeneration In all instances the volume of solvent used is 40 60 column volumes unless otherwise stated The column efficiency capacity factor etc should be measured at the start and end of the clean up procedure to ensure that it has been performed successfully and has improved the column performance Ensure that no buffers samples are present on the column and that the solvent used prior to the clean up is miscible with the first wash solvent After the clean up ensure that the test mobile phase is compatible with the last solvent in the column 5 6 1 Normal Phase Media 1 Flush with tetrahydrofuran 2 Flush with methanol 3 Flush with tetrahydrofuran 4 Flush with methylene chloride 5 Flush with benzene free n hexane 5 6 2 Reversed Phase Media 1 Flush with HPLC grade water inject 4 aliquots of 200 yL DMSO during this flush 2 Flush with methanol 3 Flush with chloroform 4 Flush with methanol 5 6 3 Anion Exchange Media 1 Flush with HPLC grade water 2 Flush with metha
4. Successful HPLC Operation A Troubleshooting Guide Version 1 1 Analyze Detect Measure Control ELECTRON CORPORATION Table of Contents 1 Introduction 1 1 Purpose 1 2 Content 2 Troubleshooting Strategy and Processes 2 1 Strategy 2 2 Troubleshooting Process 3 Troubleshooting Isolation and Corrective Action 3 1 Visual Inspection 3 2 Pressure 3 2 1 High Pressure 3 2 2 Low Pressure 3 2 3 Fluctuating Pressure 3 3 Baseline Irregularities 3 3 1 Non Cyclic Noise Fluid Path Problems 3 3 2 Non Cyclic Noise Detector Electronics Problems 3 3 3 Cyclic Noise Detector Related Problems and Others 3 4 Changes In Chromatography 3 4 1 Retention Time Changes from Injection to Injection 3 4 2 Continually Increasing or Decreasing Retention Times 3 4 3 Increasing or Decreasing to a New Constant Retention Time 3 4 4 Abnormal Peak Shape 3 5 Qualitative Results 3 6 Quantitative Results 4 Good Laboratory Practice for HPLC 4 1 Preparation of Solvents 4 1 1 Quality 4 1 2 Buffers 4 1 3 Filtration 4 1 4 Degassing 4 2 Solvent Use 4 2 1 Instrument 4 2 2 Mobile Phase Properties 4 3 Changing Solvents 4 3 1 Buffered Phase to Wash or Storage Phase 4 3 2 Normal to Reversed Phase and Vice Versa 4 3 3 General amp 5s mm S m Co N DFP A WN TD DODO DAN DD DIO MIN cO 31 31 31 31 31 32 32 32 33 33 33 33 34 4 4 System Plumbing and Fittings 4 4 1 Cutting Tubing 4 4 2 Fittings 4 4 3
5. Refer to your system maintenance manuals to determine the recommended tubing for a specific application Note Most suppliers of HPLC tubing now color code their products for ease of internal diameter identification The type of tubing used is determined by the application that is being performed The two most common types of tubing are stainless steel and PEEK although others are also available When changing tubing make sure that the replacement is manufactured from a material that is compatible with any solvents that may be flushed through it 4 4 1 Cutting Tubing Tubing should always be cut using instruments that are designed for the purpose The use of scissors and wire cutters is not recommended as they can deform the tubing bore and almost always produce angled cuts that are one of the biggest causes of leaks and increased dead volume Note Where possible pre cut tubing should always be used Stainless Steel Stainless steel can be effectively cut using the procedure outlined below 1 Estimate the length required Remember to allow extra length if the tubing is to go around corners as sharp bends in the tubing will distort the inside bore and hence the solvent flow through it 2 Using a stainless steel cutter score cleanly round the tubing If a cutter is unavailable use a knife file to score the tubing Care should be taken whichever method is used not to distort or damage the tubing 3 Using two pairs of s
6. gas Methylethylketone Phosphoric Acid 50 Sodium Hydroxide 20 Sulfuric Acid 50 Sulfur Dioxide gas Temperature C 200 200 200 200 200 200 200 200 200 PEEK Compatibility 2 5 8 USP Specifications for HPLC Columns The USP specifications are listed below with the appropriate Thermo columns listed for your convenience In some cases there is more than one column listed When in doubt it is recommended that you consult the original complete method as stated in the USP or contact our technical support team for additional information or help in choosing the correct column Phase Particle Size Particle Shape End Capped Comments L1 Octadecyl silane C18 chemically bonded to porous or ceramic microparticles 3 10 um in diameter Hypersil GOLD HyPURITY C18 HyPURITY AQUASTAR BioBasic 18 BetaBasic 18 AQUASIL C18 Hypersil BDS C18 BETASIL C18 BetaMax Neutral Hypersil ODS Hypersil ODS 2 L2 Octadecyl silane chemically bonded to a silica gel of controlled s Pellicular ODS L3 Porous silica microparticles 3 10 um in diameter BETASIL Silica Hypersil Silica 3 5 and 8 um 3 5 and 8 um 3 5 and 8 um 5 and 10 um 3 and 5 um 3 and 5 um 3 and 5 um 3 5 and 10 um 5 um 3 5b and 10 um 5 um 50 um 3 and 5 um 3 5 and 10 um spheri spheri spheri spheri spheri spheri spheri spheri spheri spheri spheri spheri spheri spheri ca yes ca yes ca ca yes ca yes ca ca yes ca
7. spheri spheri spheri spheri ca yes ca yes ca yes ca yes ca yes ca no ca yes ca no use for acids bases neutrals or chelators use for peptides proteins and other biomolecules use for basic compounds 150 appropriate for most applications 100 appropriate for most applications best for acids and neutrals best for acids and neutrals best for reversed phase applications L9 10 pm irregular or spherical totally porous silica gel having a chemically bonded strongly acidic cation exchange coating SCX Partisil SCX 10 um irregular no L10 Nitrile groups CN chemically bonded to porous silica parti HyPURITY Cyano BioBasic CN BetaBasic CN BetaMax Base BETASIL CN Hypersil BDS Cyano Hypersil CPS Hypersil CPS 2 5 um 5 um 3 and 5 um 5 um 3 and 5 um 3 and 5 um 3 5 and 10 um 5 and 10 um spheri spheri spheri spheri spheri spheri spheri spheri ca yes ca yes ca yes ca yes ca yes ca yes ca no ca yes 80 pore size cles 3 10 um in diameter use for acids bases neutrals or chelators use for peptides proteins and other biomolecules 150 appropriate for most applications very retentive 100 appropriate for most applications use for basic compounds use for acids and neutrals better for reversed phase applications 57 Phase Particle Size Particle Shape End Capped Comments L11 Phenyl groups chemically bonded to porous silica microparticles 5 10
8. 5 1 5 Selectivity a Selectivity is a measure of the relative retention of two adjacent peaks in a chromatogram It can be calculated using capacity factors or retention volumes Et EN g k Vi Vo Where k V capacity factor ky V capacity factor V void volume V peak 1 retention volume V peak 2 retention volume Selectivity can be affected by changes in mobile phase composition temperature and column chemistry Changes in selectivity that occur both with standard and sample mixes are most likely to be due to changes in the column temperature or mobile phase composition Changes in selectivity that occur only in the sample mix and not the standard mix are most likely to be due to the composition of the sample 5 1 6 Band Spreading or Band Broadening Broad peaks often accompanied by a change in retention time indicate band spreading It can occur within the HPLC column but is more often due to incorrect system plumbing The following procedure describes a method for measuring the band spreading due to the HPLC system Column effects can be measured using efficiency calculations 1 Remove the HPLC column from the system and replace with a zero dead volume union 2 Configure the HPLC system with the following parameters Flow Rate 1 mL min Detector Sensitivity 0 5 to 1 0 AUFS Detector Time Constant 0 2 or less Chart Speed if req d 20 cm min 3 Perform a ten fold dilution of the column efficiency tes
9. 7 5 1 3 Tailing Factor T Tailing factor is a measure of the symmetry of a peak Ideally peaks should be Gaussian in shape totally symmetrical A peaks tailing factor is measured using the following equation Where Woo peak width at 5 height f distance from peak front to apex point at the baseline f Wo os 43 5 1 4 Capacity Factor k Capacity factor is a measure of the retention of an analyte relative to the column void volume Vo It is measured using the following equation Vi Vo k a Vo Where V Column void volume V Retention volume of peak Important The column void volume can be measured by injecting a compound that will be unretained by the column packing Typical compounds used include uracil RP and toluene NP Contact your column supplier for advice if you are unsure about the retention characteristics of your chosen void marker Capacity factor is affected by changes in mobile phase operating temperature analyte retention characteristics and changes to the surface chemistry of the column Changes in capacity factor that occur both with standard and sample mixes are likely to be due to changes in the column temperature or mobile phase composition Changes in capacity factor that occur only in the sample mix and not the standard mix are most likely to be due to the composition of the sample Note Capacity factor will change by up to 10 for a 5 C rise in column temperature
10. SLIPFREE Fittings 4 4 4 Blocked Tubing 4 4 5 Old and Leaking Fittings 4 5 Column Selection 4 6 Column Protection 4 6 1 Guard Columns and Cartridges 4 6 2 Pre Column Filters 4 7 Column Operating Temperature 4 8 Sample Preparation 5 Reference Data 5 1 5 1 5 Chromatographic Performance Tests 5 1 6 Band Spreading or Band Broadening 5 2 System Calculations 5 2 1 Column Backpressure and Typical Operating Flow Rates 5 2 2 Scaling Up or Scaling Down 5 3 Solvent Properties 5 4 Buffer Properties 5 5 Chromophore Detection Wavelengths 5 6 Column Cleaning and Regeneration 5 6 1 Normal Phase Media 5 6 2 Reversed Phase Media 5 6 3 Anion Exchange Media 5 6 4 Cation Exchange Media 5 6 5 Protein Size Exclusion Media 5 6 7 Porous Graphitic Carbon 5 6 8 Polymeric Media with Metallic Counter lons 5 7 Chemical Compatibility of Polymeric Tubing With Solvents 5 8 USP Specifications for HPLC Columns Appendices Appendix A Column Selection Guide Appendix B Solvent Miscibility Chart Appendix C Example Routine Maintenance Log Appendix D Example Problem Log Appendix E Example Column History Log Appendix F Example Instrument Use Log References 34 35 36 37 37 38 38 39 39 40 40 41 42 42 45 46 46 4 48 49 51 52 52 52 52 52 52 53 54 55 57 60 63 64 65 66 67 68 1 Introd 1 1 Purpose 1 2 Content uction This guide is intended as an aid in troubleshooting problems that occur in the day
11. better protein and peptide separations e Excellent reproducibility efficiency and column lifetimes Range of stationary phases to optimize selectivity including ion exchange and size exclusion e Ideal for proteomics and LC MS of biomolecules e Excellent alternative to Vydac YMC Pack and Zorbax wide pore columns Performance Columns BetaBasic Columns Stable inert and reproducible for general HPLC Superb stability at both high and low pH Efficient and reproducible columns A family of useful stationary phases 150A pore size for small molecules peptides and protein digests Excellent alternative to YMCbasic columns BETASIL Columns High retention efficiency and selectivity Highly retentive phases with excellent peak shapes and lifetimes High surface area with high bonded phase coverage Phases with unique selectivity for reversed phase and normal phase deal for LC MS applications including short fast DASH columns Excellent substitute for Inertsil columns BetaMax Columns Maximum retention for maximum results Ultra high surface area and surface coverage for maximum retention 3 chemistries for 3 types of analytes acid base and neutral Excellent stability for long column lifetimes BetaMax Neutral provides maximum C18 reversed phase retention for LC MS applications Classical Hypersil Columns Classical Hypersil reversed phase chemistries from Thermo provide a proven record of performan
12. between tightened fittings replace the fitting and ferrule since they may be damager or misaligned 3 4 3 Increasing Decreasing to a New Constant Retention Time Possible Cause Mobile Phase Inconsistencies Changes in Solvent Flow Rate Incorrect Column Temperature Changes Column Contamination Gradient Delays Corrective Action Check that the solvents in use are the correct ones of the correct strength and are present in the correct composition If additives for example preservatives are used ensure that they are the correct ones and do not interfere with the analyte in your sample Prepare fresh solvents Note It is always worth checking the pH of any buffers used If the buffer is not freshly prepared then it may have been contaminated or absorbed CO from the atmosphere etc These factors would affect the buffer pH and ultimately your sample chromatography Ensure that the flow rate delivered is the same as that entered into the pump software Check all solvents lines that are in use for the method If the measured flow rate is not the same as that entered into the pump software refer to the pump maintenance manual for further assistance or contact your maintenance provider Also ensure that the flow rate being used is correct for the application Check that the column in place in the system is the correct one for the method Dimensions and packing materials will make a considerable differ ence between chromat
13. in reverse flow mode Pump in reverse flow mode Pump in reverse flow mode at 0 1 mL min with 0 1M Ca NOs at 0 1 mL min with 20 80 at 0 1 mL min with 20 80 pH 6 3 and 85 C for 4 to 16 hrs ACN H 0 25 C for 4 hrs ACN H 0 25 C for 4 hrs Sodium Counter lon Pump in reverse flow mode Pump in reverse flow mode Pump in reverse flow mode at 0 1 mL min with 0 1M NaNO at 0 1 mL min with 20 80 at 0 1 mL min with 20 80 85 C for 4 to 16 hrs ACN H 0 25 C for 4 hrs ACN H 0 25 C for 4 hrs Silver Counter lon No Regeneration Pump in reverse flow mode Pump in reverse flow mode Procedure Available at 0 1 mL min with 20 80 at 0 1 mL min with 25 80 ACN H 0 25 C for 4 hrs ACN H 0 25 C for 4 hrs Lead Counter lon Pump in reverse flow mode Pump in reverse flow mode Pump in reverse flow mode at 0 1 mL min with 0 1M Pb NO at 0 1 mL min with 20 80 at 0 1 mL min with 20 80 pH 5 3 and 85 C for 4 to 16 hrs ACN H 0 25 C for 4 hrs ACN H 0 25 C for 4 hrs 5 7 Chemical Compatibility of Polymeric Tubing with Solvents The first table in this section contains information on the compatibility of a wide range of solvents at 20 C with PEEK polyethylene polypropylene PVDF Teflon and Tefzel The second table contains information on the compatibility of PEEK with solvents at elevated temperature Note PEEK tubing will swell in the presence of DMSO methylene chloride and THF Solvent Compatibility with Polymeric Tubing
14. is incorrect Inject the correct volume Zero the detector output Check the output signal between the detector and data handling device If the detector flow cell is contaminated clean the flow cell windows as per the instructions in your detector maintenance manual Check that the detector lamp energy is within specified limits for operation If the energy is markedly lower than when the lamp was new replace it Note Some models of detector allow the lamp energy level to be manipulated when the lamp nears the end of its useful lifetime Refer to your detector maintenance manual for details if applicable Finally ensure that the detector wavelength setting is optimized for the analyte chromophores Refer to section 5 5 of this guide for chromophore detection wavelengths Dilute the sample or decrease the rate at which the syringe draws the sample Early Eluting Peaks Broad J Normal Chromatography Abnormal Chromatography Possible Cause Sample Overload Blockage Before the Column Associated Pressure Increase Incorrect Tubing ID Injector Problem Detector Time Constant Incorrect Late Eluting Peaks Carry over Corrective Action Dilute the sample or inject a lower volume to stop equilibrium disruption Note Using a weaker solvent means that the injection volume can be increased to approximately 10 of the column void volume Using a strong solvent means that the injection volume can
15. left The effect of solvent heating prior to analysis is shown below Without Eluent Pre Heater With Eluent Pre Heater 80 C 80 C oven3 oven4 O0 1 2 3 4 5 MIN 0 1 2 S3 4 5 MIN Kromasil C8 5 pm 200 x 4 6 mm Sample Enalapril Maleate Part No 16205 204630 Eluent 30 ACN 70 0 03M KH PO pH 4 0 Flow 1 8 mL min Detector UV 215 Data courtesy of Dr Richard F Myer Quantitative Tem perature 80 C Technologies Inc Whitehouse NJ The recommended operating temperatures for columns is very much dependent on the analytical conditions and the packing inside the column Typically carbon and polymer based columns can be used at far higher temperatures than can a silica based column This is because elevated temperatures increase the risk of chemical attack on the bonding and base silica by the solvent For example at 60 C and with a buffer at pH 2 a silica based amide column packing can start to hydrolyze This would drastically affect the chromatography available from the column The choice of particle size is also a contributing factor when considering operating temperature Particles below 5 um in diameter are more prone to bed collapse and loss of efficiency at highly elevated temperatures Temperature can be a powerful method development tool if it is used correctly Lower temperatures can be used to help increase retention selectivity and resolution Higher temperatures can be used to h
16. measure exact quantities of an analyte in solution Therefore problems associated with these assays usually fall into one of two categories a loss of precision and b loss of accuracy The most common mistake made is to assume that accuracy and precision are the same Accuracy is defined as the proximity of a result to the true value whereas precision is a measure of reproducibility of the result It is important to determine whether your method is inaccurate or imprecise as this will determine the steps taken to rectify the problem In general if the result generated is incorrect but reproducible then the problem is accuracy based If the results generated vary then the problem is precision based The following tables will assist in tracing errors in quantitative methods Loss of Accuracy Possible Cause Corrective Action Incorrect Sample Sample Preparation Check that the correct sample has been prepared and that the preparation has produced a solution that is of the correct strength At the same time check that any internal external standards have been prepared correctly and are of the correct strength Solvent Evaporation Samples prepared in volatile solvent such as chloroform DCM etc are affected by changes in ambient temperature They will evaporate more quickly in warmer temperatures producing more concentrated sample solutions Ensure that the ambient temperature around the sample remains constant and that the vial cap is
17. only be up to 1 of the column void volume Check the guard in line filter column inlet and all associated tubing or blockages Replace any blocked tubing filters or guard units If the column inlet frit has been blocked gently back flush the column with a wash solvent at very ow flow rate preferably over night for best effect Re invert the column and equilibrate with test solvent If this procedure has not removed the blockage please contact your column supplier for further technical advice Measure the level of band spreading throughout the system Refer to section 5 1 6 of this guide for details of the calculation If the level of band spreading is greater than the limits specified for the system ensure that all tubing is appropriate for the job it is intended for for example all internal diameters are as narrow as possible Also check that all tubing is cut with a flat end This will enable it to fit into unions or fittings with far greater efficiency and minimize excess dead volume With reference to the autosampler maintenance manual check that the valves in the injector system are not sticking or leaking Clean or replace as necessary Also check that the needle and seat are not blocked or damaged Once again clean or replace as necessary Determine the detector time constant setting and adjust accordingly Details of how to determine the time constant can be found in the detector operator manual Ensure that
18. please contact columninfo uk thermo com www thermo com chromatography ANSI RAB ES Bal QMS 01110342 UKAS ISO 9001 2000 MANAGEMENT FM 76231 013 Thermo Electron Corporation Thermo Hypersil Ltd Bellefonte PA is ISO Certified Runcorn UK is ISO Certified 2004 Thermo Electron Corporation All rights reserved Specifications terms and pricing are subject to change Not all products are available in all countries Please consult your local sales representative for details TG20094 E 10 04M ELECTRON CORPORATION
19. sample Inject a more concentrated sample solution or a larger volume of the same sample solution Incorrect injection volume Inject the correct volume of sample Needle blocked Remove the injector needle from its support as per the autosampler maintenance manual and either clean or replace it The blockage may be due to the vial septa degrading or coring Ideally replace all vial septa caps with a non coring variety Other sources of blockage include particulates in the sample Ensure that all samples are filtered prior to injection Typically no flow or very low flow Ensure that there is power to the pump and that it is switched on Check that solvent comes out of the detector waste line when the pump is set running Check that the solvent reservoirs contain solvent and that the solvent inlets are at a suitable height to draw any liquid present Also check that the fluid inlet filters and lines are not blocked If no solvent flow occurs refer to the pump maintenance manual or contact your maintenance provider Typically incorrect or missing column Check that there is a column in the system that it is the correct dimension and that it is packed with the correct media for the application Ensure that the detector is set to the correct wavelength and that sensitivity and auto zeros are also correctly set Zero the detector baseline if necessary Check all power cables and connections between the detector and data handling dev
20. the problem lies within the fluid path perhaps with incorrect ID tubing fittings etc If single peaks are affected then it suggests that there might be a specific chemistry problem The method in use should be examined for areas where the chemistry may not be correct Gradient methods where early eluting peaks are abnormal and later peaks are acceptable may be suffering from pre column band broadening If all the peaks are abnormal then post column band broadening or other changes in the system are the most likely causes Isocratic methods where the early eluting peaks are abnormal and the later peaks are acceptable may be suffering from extra column band broadening injector problems incorrect detector time constant or incorrect A D sampling time If all the peaks are abnormal then extra column band broadening or other changes to the system are the most likely causes Each table in this section will show an example of the type of chromatography being investigated it s cause s and any corrective action that can be taken No Peaks Possible Cause Injector Problem Fluid Path Problem Column Problem Detector Problem Incorrect Solvent Sample Corrective Action No vial Fill autosampler position with a vial Empty vial Fill the vial with sample Over full vial Replace with a vial where there is an air gap between the top of the fluid and vial cap Wrong vial Check that the correct sample was injected Insufficient
21. to day running of your HPLC system It is also intended as a guide to best practice in systems that are free from problems Its primary aims are to provide a logical process when troubleshooting to maximize system operation time and to promote good chromatographic practices Wherever possible diagrams or schematics are used for clarity This enables the guide to be used quickly and efficiently by operators with varying experience Quick Tips at the end of each section are included for rapid troubleshooting and less experienced HPLC users Each troubleshooting chapter contains information on the most likely cause s of the observed problem and ways in which they can be rectified Each table has the most probable cause of the observed fault as it s first entry The scope of this guide has been extended where necessary to include LC MS column and instrument troubleshooting The focus of the bulk of the causes of problems and their corrective action is the UV and other flow through detectors 2 Troubleshooting Strategy And Processes 2 1 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 Returning the unit to routine use OR referring the problem to your maintenance manager 2 2 Troubleshooting Process To execute the strategy a systematic approach which will work f
22. yes ca yes ca yes ca yes Ca no Ca E Ca use when peak shape is critical use for acids bases neutrals or chelators wettable packing for enhanced polar retention use for peptides proteins and other biomolecules appropriate for most applications use with highly aqueous eluents use for basic compounds 100A for high retention use with at least 10 20 organic eluents best for acids and neutrals alternative to Waters Spherisorb ODS 2 columns urface porosity bonded to a solid spherical core 30 50 pm in diameter available in 60A 100A 200A and 300A general purpose L4 Silica gel of controlled surface porosity bonded to a solid spherical core 30 50 um in diameter Pellicular Silica 50 um spheri Ca z L5 Alumina of controlled surface porosity bonded to a solid spherical core 30 50 um in diameter L6 Strong cation exchange packing sulfonated fluorocarbon polymer coated on a solid spherical core 30 50 um in diameter L7 Octyl silane C8 chemically bonded to porous or ceramic microparticles 3 10 ym in diameter HyPURITY C8 BioBasic 8 Hypersil BDS C8 BetaBasic 8 BETASIL C8 Hypersil MOS Hypersil MOS 2 L8 An essentially monomolecular layer of aminopropylsilane chemically bonded to totally porous silica gel support 10 pm in diameter Hypersil APS 2 3 and 5 um 5 um 3 and 5 um 3 and 5 um 3 and 5 um 3 5 and 10 um 3 5 and 10 um 3 5 and 10 um spheri spheri spheri spheri
23. 49 0 007 0 632 0 25 0 507 0 010 1 288 0 51 2 026 0 020 5 146 1 02 8 103 0 040 20 581 45 5 2 System Calculations 5 2 1 Column Backpressure and Typical Operating Flow Rates Column operating backpressure is affected by column length internal diameter media particle size temperature solvent properties and solvent flow rate It can also be affected by the use of gradients where the pressure may vary with solvent composition Typical operating backpressure for columns or cartridges can be calculated quite easily The initial calculation uses water as the mobile phase The backpressure obtained is then multiplied by the viscosity of the actual mobile phase The calculation is Pressure at 1 mL min water 21 x f xL x n dp x d Where Pressure is measured in atmospheres f 1000 for columns 4 6 mm ID columns L Column length cm dp Particle diameter mm d Column diameter mm n Mobile phase viscosity centipoises Mobile phase viscosity varies with composition As an example the following plot shows how water viscosity varies with the addition of methanol or acetonitrile 2s 4H Acetronitrile Methanol 2 1 1 8 1 5 1 2 0 9 Viscosity cp 0 6 0 3 0 T T T T T T T 0 10 20 30 40 50 60 70 80 90 100 Water in Mixture Note Although HPLC columns are manufactured under extremely high pressure it is recommended that you do not exceed the maximum pressure recommended by your instru
24. 8 9 Time Minutes Time Minutes Mobile Phase Not Degassed Mobile Phase Degassed The most efficient form of degassing is bubbling with helium or another low solubility gas If this method is available we recommend that the mobile phase is continually degassed at very low levels throughout the analysis This will inhibit the re adsorption of gases over the analysis time Note Ensure that the solvent reservoir has a vent to the atmosphere to prevent the build up of pressure inside the reservoir 4 2 Solvent Use 4 2 1 Instrument Each solvent line should be fitted with an inlet filter This is the first line of system defence against particulate contamination from solvents The filters should be kept clean to prevent cross contami nation When they are not being used it is recommended that they are stored in a solution of 50 acetonitrile 50 water This will inhibit microbial growth and stop dust and dirt from embedding in the filter pores The solvent lines should be clean growth free and should have no sharp bends or creases in them Solvent reservoirs should be placed as high as possible on or in the instrument always higher than the pump inlet manifold The solvent lines and filters should be of sufficient length to reach the bottom of the solvent reservoir Important Before starting an analysis calculate the total volume of mobile phase required Prepare all the phases at the same time and place in reservoirs th
25. Glycine Hydrochloride Glycine 8 8 10 8 9 8 Glycylglycine 84 HEPES N 2 Hydroxyethylpiperazine N 2 ethanesulfonic Acid 7 0 8 0 7 55 HEPPS 76 86 8 00 MES 2 N Morpholino ethanesulfonic Acid 5 8 6 5 6 15 1 Methylpiperidine Hydrochloride 1 Methylpiperidine 91 111 10 1 MOPS 6 5 7 9 7 20 Phosphoric Acid Mono amp Di potassium phosphate lt 3 1 2 1 6 2 8 2 7 2 11 3 13 3 12 3 PIPES Piperazine N N Bis 2 ethanesulphonic Acid 64 72 6 8 TAPS N Tris hydroxymethyl methyl 3 aminopropanesulfonic Acid 7 4 94 84 TES N Tris hydroxymethyl methyl 2 amino ethanesulfonic Acid 7 0 8 0 7 5 Trifluoroacetic Acid 1 5 2 5 gt gt 2 Tricine N Tris hydroxymethyl methylglycine 76 88 8 15 Triethylamine Hydrochloride Triethylamine 10 0 12 0 11 0 TRIS Tris hydroxymethylJaminomethane 73 93 8 3 49 Buffer choice will be very dependent on the analyte and the instrumentation used Ideally LC MS applications should use a volatile buffer as this will not form a contaminating deposit on the cone and source Inorganic acids involatile buffers and ion pair reagents should all be avoided Typical LC MS buffers include Ammonium acetate formate hydrogen carbonate lt 50mM Formic acetic acid 0 01 1 v v Trifluoroacetic acid 0 196 v v Trialkylamine and aqueous ammonia type bases TRIS BIS TRIS propane Electrolyte additives are often added to LC MS buffers to improve peak shape These additives should also be volatile Car
26. ITY AQUASTAR Columns amp AQUASIL C18 Columns Retention of polar compounds Polar end capped reversed phase columns for enhanced retention of polar compounds Compatible with 10096 aqueous mobile phases and standard reversed phase mobile phases Run with reduced buffer concentrations e C18 phase for reliability and high performance e Ideal alternative to YMC Pack ODS AQ Phenomenex AQUA MetaSil AQ and other 2nd generation polar end capped columns Fluophase Columns Fluorinated phases for highly selective reversed phase chromatography Range of fluorinated phases provides unique selectivity Extra retention and selectivity for fluorinated and chlorinated compounds Excellent selectivity for polar non halogenated compounds with nitro hydroxyl carboxyl and other polar groups e Shape selective for structural isomers Use typical reversed phase solvents and conditions Fluophase RP amp WP Fluophase PFP HyPURITY ADVANCE Columns amp PRISM Columns Polar embedded phase for reversed phase chromatography Unique reversed phase selectivity End capped and non end capped versions of PRISM for selectivity choices deal for the separation of polar compounds Excellent alternative to Waters SymmetryShield Zorbax Bonus RP or Supelcosil ABZ Plus columns d Polar Functionality Appendix B Solvent Miscibility Chart Acetic Acid Acetone
27. REZ XP Carbohydrate H 8 um spherical use for organic acids alcohols glycoproteins etc L18 Amino and cyano groups chemically bonded to porous silica particles 3 10 um in diameter L19 Strong cation exchange resin consisting of sulfonated cross linked styrene divinylbenzene copolymer in the calcium form about 9 pm diameter HyperREZ XP Carbohydrate Ca 8 um spherica 7 use for saccharides alcohols food adulteration etc L20 Dihydroxypropane groups chemically bonded to porous silica particles 5 10 um in diameter BETASIL Diol 100 5 um spherica yes 100A appropriate for most applications L21 A rigid spherical styrene divinylbenzene copolymer 5 10 pm in diameter HyperREZ XP RP100 5 um spherica yes 100A for compounds needing pH extremes and high temperature HyperREZ XP RP300 5 um spherica yes 300A for peptides and small proteins L22 A cation exchange resin made of porous polystyrene gel with sulfonic acid groups about 10 pm in size HyperREZ XP SCX 8 um spherica yes 1000A for macromolecules L23 An anion exchange resin made of porous polymethacrylate or polyacrylate gel with quaternary ammonium groups about 10 pm in diameter L24 A semi rigid hydrophilic gel consisting of vinyl polymers with numerous hydroxyl groups on the matrix surface 32 63 pm in diameter L25 Packing having the capacity to separate compounds with a MW range from 100 to 5000 daltons as determined by polyethylene oxide applied to neutral anio
28. age between the syringe needle and detector Problems with the syringe plunger sticking in the barrel can occur if the sample contains particulates Note Viscous samples will require a longer draw time Insufficient draw time will result in a lower volume of sample being injected onto the column and smaller peaks will result l Quick TIP Smaller Than Expected Peaks Normal Chromatography 0 Abnormal Chromatography Possible Cause Vial Problem Syringe Malfunction Sample Loop Incorrect Wrong Injection Volume Detector Problem Sample Too Viscous Corrective Action Check that the vial contained enough of the correct sample to perform the injection if not replace with a fresh one Make sure that the vial seats correctly in the autosampler and that the needle is not obstructed when performing an injection Note If the vial were over full a vacuum would form as the needle tried to draw solvent from it This would result in smaller than normal peaks Ensure that all sample vials contain up to two thirds liquid and one third air and that the caps where possible are not over tightened Check the syringe for cracks dirty or worn barrels and plungers sticking etc Remove and clean the syringe where possible Where this is not possible remove and replace Check the needle for blockages and deformities Replace where necessary Change the sample loop to the correct volume if the one in situ
29. ality HPLC grade reagents may cost slightly more than lower grade reagents but the difference in purity is marked HPLC grade reagents contain no impurities to produce spurious peaks in a chromatogram baseline whereas AR grade reagents do contain trace levels of impurity which may produce spurious baseline peaks Ensure that any water used in buffer preparation is of the highest purity Deionized water often contains trace levels of organic compounds and so therefore is not recommended for HPLC use Ultra pure HPLC water 18MQ resistivity is generated by passing deionized water through an ion exchange bed Modern water purification instruments use this mechanism to produce water of suitable quality in high volumes Alternately HPLC grade water can be purchased from solvent suppliers Important Do not store HPLC grade water in plastic containers Additives in the plastic may leach into the water and contaminate it Always store HPLC grade water in glass containers 4 1 2 Buffers All buffers should be prepared freshly on the day required This practice ensures that the buffer pH is unaffected by prolonged storage and that there is no microbial growth present Changes in pH and microbial growth will affect chromatography If buffer solutions are stored be aware that they have a finite lifetime Refer to pharma copoeia monographs or similar for further guidance on buffer shelf life Buffer reagents can contain a stabilizing agent for example s
30. ample solvent sample and mobile phase They are also caused after routine maintenance when the system has not been reconfigured correctly Negative Peaks Normal Chromatography Abnormal Chromatography Possible Cause Signal Polarity Setting Incorrect All Peaks Cables Reversed All Peaks RI Detector Only Unbalanced Optics All Peaks Highly Adsorbing Mobile Phase 21 Peak Air Bubble Injected in System 21 Peak RI of Analyte Lower than that of the Mobile Phase 21 Peak lon Pair Separation Only System Peak 21 Peak Corrective Action Change the polarity setting and repeat the injection The peaks should automatically invert Refer to the detector operator manual for further details Reverse the cables from the detector to the data handling device Balance the optics Refer to the detector operator manual for further details Dissolve the sample in mobile phase If the peak is due to contaminated solvents replace all solvents Refer to the injector system maintenance manual for troubleshooting Determine whether the peak is due to sample or solvent contamination If the peak is due to contaminated solvents replace all solvents Dissolve the sample in mobile phase 3 5 Qualitative Results Qualitative assays do not measure exact quantities of an analyte in solution Therefore problems associated with these assays usually fall into one of two categories a missi
31. and exchange packing L proline copper complex covalently bonded to irregularly shaped silica particles 5 to 10 um in diameter L33 Packing having the capacity to separate proteins by molecular size over a range of 4 000 to 400 000 daltons It is spherical silica based and processed to provide pH stability BioBasic SEC 5 um spherical 60 120A 300A and 1000 for aqueous size exclusion L34 Strong cation exchange resin consisting of sulfonated cross linked styrene divinylbenzene copolymer in the lead form about 9 um in diameter HyperREZ XP Carbohydrate Pb 8 um spherical use for saccharides food adulteration etc L35 A zirconium stabilized spherical silica packing with a hydrophilic diol type molecular monolayer bonded phase having a pore size of 150A L36 A 3 5 dinitrobenzoyl derivative of L phenylglycine covalently bonded to 5 pm aminopropyl silica L37 Packing having the capacity to separate proteins by molecular size over a range of 2000 to 40 000 daltons It is a polymethacrylate gel 138 A methacrylate based size exclusion packing for water soluble samples L39 A hydrophilic polyhydroxymethacrylate gel of totally porous spherical resin L40 Cellulose tris 3 5 dimethylphenylcarbamate coated porous silica particles 5 20 um in diameter L41 Immobilized a1 acid glycoprotein on spherical silica particles 5 um in diameter L42 Octylsilane and octadecylsilane groups chemically bonded to porous silica partic
32. at are large enough to accommodate them Insufficient mobile phase may cause the system to pump dry This is undesirable because it will fill the system with air If this does happen purge the solvent lines and pump with fresh solvent then allow the system to pump solvent until it is equilibrated 4 2 2 Mobile Phase Properties Do not use highly acidic or basic solvents unless your HPLC system and column have been engineered to accommodate them Seals plungers etc can be damaged by extreme pH conditions If in doubt please contact your column or instrument supplier before using an aggressive solvent The use of highly aqueous mobile phases is becoming more popular as safety guidelines demand less exposure to organic solvents Care should again be taken that the HPLC column has been engineered to accommodate highly aqueous solvents traditional alkyl chain media can be prone to phase collapse in low organic composition solvent mixes for example at less than 5 organic solvent Highly aqueous mobile phases are ideal breeding grounds for microbes Ensure that an organic solvent is flushed through the HPLC system and column at least once every 48 hours to kill unwanted microbial growth Alternatively add a small amount of sodium azide to the aqueous solvent to inhibit growth Note Never allow a HPLC column or system to stand with water or buffer in it for an extended period of time for example over a holiday break Always flush with a solv
33. at the flow rate delivered is the same as that entered into the pump software Also ensure that the flow rate being used is correct for the application Refer to the previous table Refer to the previous table Using a standard mixture solvent and analysis conditions as close to those used to generate your column s test certificate as possible calculate the columns efficiency N If N is markedly lower than quoted on the manufacturers certificate of analysis try cleaning the column using the procedures in section 5 6 of this guide Repeat the efficiency calculation If there is no increase in efficiency replace the degraded column with a new one You may wish to contact your column supplier if the degraded column is relatively new to discuss whether it was a handling error that caused the degradation Ensure that all solvents are freshly prepared and free from microbial growth Filter and degas thoroughly prior to use Discard old solvents and thoroughly wash all dirty reservoirs prior to re use Prime the pump and solvent lines with freshly prepared solvents and allow the column system to equilibrate Check the solvent lines and inlet filters for blockages Refer to the pump maintenance manual for cleaning replacement of blocked or dirty parts Check all fittings and unions for leaks Tighten any loose fittings but do not be tempted to over tighten as this may damage the fitting s threads and cause leaks Where leaks occur
34. ce for routine analyses year after year Specified in thousands of methods world wide our classical Hypersil families offer exceptional reproducibility and reliability Hypersil BDS Columns The standard base deactivated column e Base deactivated for reduced peak tailing and excellent peak symmetry Highly reproducible and efficient Reliable with long column lifetimes Excellent performance with basic acidic and neutral compounds Hypersil Classical Columns Reliable and reproducible Excellent phase used for thousands of existing methods High efficiency proven reproducibility and long column lifetimes Wide range of bonded phases including dedicated columns for specific applications 61 Unique Selectivities Thermo offers a variety of HPLC columns with unique selectivities for both routine and challenging separations These columns provide chromatographic solutions for many problematic analyses Choose any of our innovative phases for method development or for standard separations Please contact Technical Support for assistance in choosing the appropriate column for your application Hypercarb Columns 100 porous graphitic columns solve problem separations e Exceptional retention of polar compounds pH stable from 1 to 14 Separate structurally similar compounds deal for LC MS applications Superior performance compared to Waters XTerra ZirChrom and other non silica based columns HyPUR
35. coated porous spherical silica particles 5 10 um in diameter L52 A strong cation exchange resin made of porous silica with sulphopropyl groups 5 10 yum in diameter BioBasic SCX 5 um spherical z 300 strong cation exchanger for peptides and proteins 59 Appendix A Column Selection Guide Thermo Column Hydrophobicity Chart BetaMax Neutral BETASIL C18 Kromasil C18 Symmetry C18 Columbus Genesis C18 YMC ODS Inertsil ODS 2 Zorbax ODS AQUASIL C18 YMC AQ BetaBasic 18 Hypersil BDS C18 Hypersil ODS KEYSTONE PAH BETASIL C8 Kromasil C8 Luna C8 LiChrosorb C18 Hypersil PAH HyPURITY C18 Waters Spherisorb ODS 2 BETASIL C6 PRISM RP Waters Spherisorb C8 SynChropak RP P 100 Waters Spherisorb ODS 1 Supelcosil LC ABZ BioBasic 18 Luna Phenyl hexyl Hypersil MOS 2 HyPURITY AQUASTAR BetaBasic 8 Hypersil BDS C8 HyPURITY C8 Kromasil C4 PRISM RPN YMC Basic Zorbax Stablebond C8 VYDAC 201HS C18 DELTABOND ODS BetaBasic 4 DELTABOND Octyl BioBasic 8 BETASIL C1 BETASIL CN BETASIL Phenyl BioBasic 4 Fluofix 120 BetaBasic Phenyl Hypersil Phenyl Hypersil BDS Phenyl HyPURITY ADVANCE BetaBasic CN DELTABOND CN Very Retentive ot __t_ Ja BetaBasic 18 5um 150x4 6mm Eluent 75 ACN 25 H O Flow 1 25 mL min Detector UV 254 Sample 1 Uracil Benzene C2 substituted benzene C3 substituted benzene C4 substituted benzene C5 substituted benzene C6 substituted benzene C7 sub
36. d at x height Ti B Distance from apex to peak start at x height x Percentage of height h at which efficiency is measured 5 1 2 Column Efficiency N Column efficiency or the theoretical plate count is a measure of peak band spreading The lower the level of band spreading the higher the column efficiency and vice versa Important The column efficiency figure quoted on the supplied certificate of analysis is actually the efficiency for the column AND HPLC system If the efficiency calculation is repeated on a different instrument when the column is new it is very likely that there will be a difference between the certificated value of N and your new calculated value of N This difference is not due to the column but the instrument There are a number of different methods used to calculate column efficiency Some take into account peaks that are unsymmetrical others do not For consistency the method you use should always be the same In order of sensitivity to peak shape most sensitive first the calculations for measuring N are as follows a Asymmetry Based N 412 TW 1 25 A B Where x 10 b 5 Sigma N 25 T W 2 Where x 4 4 c 4 Sigma N 16 T W Where x 13 4 d Tangent N 16 T W W d Tangent Refer to diagram on right for W e 3 Sigma N 9 T W Where x 32 4 f Half height N 5 54 T W 2 Where x 50 g 2 Sigma inflection N A T WE Where x 60
37. d of time for equilibration must be allowed between gradient Gradient Analysis analyses This allows the T mobile phase composition to be pumping through the column as the injection occurs Insufficient equilibrium time results in erratic retention times Pump Pressure Action Problems Refer to the flow chart in section 3 2 3 of this guide Identify the problem and correct it Ambient Temperature Variations Stabilize the ambient temperature around the instrument and allow it to come to equilibrium If this is not possible or cannot be achieved we recommend the use of a column heater cooler The unit should fully enclose the column and ideally be adjustable to compensate for multiple column dimensions Ensure that all pre mixed solvents are miscible and that the solution is homogeneous Note Some methods will also benefit from pre heated solvents prior to passage through the column Volume Injection Concentration Too High Either reduce the injection volume used or dilute the original sample Note Using a weaker solvent means that the injection volume can be increased to approximately 10 of the column void volume Using a strong solvent means that the injection volume can only be up to 1 of the column void volume 3 4 1 Retention Time Changes from Injection to Injection Continued Possible Cause Solvent Blending Problems Column Contamination Corrective Action Check the miscibility of the solvents if their miscibilit
38. d units are also available for LC MS and use with finger tight fittings Please contact your Thermo HPLC columns representative for more information Diameter mm Column Guard 1 1 P 2 and 2 1 2 3 3 3 9 and 4 4 46 4 or 4 6 78 78 10 10 39 Solvent Heater Unit 4 6 2 Pre Column Filters These units are positioned between the solvent inlet filter and the column inlet They are designed to trap particulates from the fluid path They will not trap dissolved substances Once again these units are designed to be disposable and should be replaced regularly There are a number of designs of filters available For further information on these designs or for advice on your application please contact your Thermo representative 4 7 Column Operating Temperature It is vitally important that columns are maintained at a constant temperature while in use Fluctuations in temperature can cause peak drift and other undesirable effects in your chromatography For this reason we recommend the use of a thermostatically regulated temperature control unit to house your column during use such as HOT POCKET or COOL POCKET temperature controllers An ideal temperature control unit will both cool and heat the column efficiently and is not subject to changes in the ambient room temperature In applications where the column will be maintained at temperatures above or below ambient it is also worth considering the use of a solvent heater shown on the
39. der and perform an injection If the problem remains it is not related to the guard If the problem disappears check the connections between the guard unit and the column Ensure that the connector adds zero dead volume to the system and that the guard cartridges fit snugly into the holder Ideally use an integral guard Dissolve the sample in mobile phase use a weaker diluent or make a smaller injection The most common cause of peak doubling can be either blockage prior to the column or column or guard voiding 23 Fronting peaks are very often due to large injection volumes of a sample that is dissolved in solvents that are incompatible with the mobile phase being used The next most common cause of peak fronting is a voided or contaminated guard or column Fronting Peaks o J o ES Normal Chromatography Abnormal Chromatography Possible Cause Injection Disrupting Equilibrium Column Voiding Guard Column Degrading and Contamination Corrective Action Dissolve the sample in mobile phase use a weaker diluent or make a smaller injection Using a standard mixture solvent and analysis conditions as close to those used to generate your column s test certificate as possible calculate the columns efficiency N If N is markedly lower than is quoted on the manufacturers certificate of analysis try cleaning the column using the procedures in secti
40. e baseline for 5 to 10 minutes Note if there is any improvement in the baseline s appearance If yes then the problem lies within the instrument fluid path If no the problem is either electrical or detector related 3 Disconnect the detector electrical cables from the A D interface with the PC integrator and chart recorder i e the data handling devices Attach a jump source to the input terminals on the data handling device a crocodile clip paper clip etc If the noise ceases then the problem is within the detector or it s electrical connections If the noise continues then the problem is within the data handling device Data handling device troubleshooting is beyond the scope of this guide We recommend that you contact your instrument provider for this service The sections provide a quick reference guide for typical baseline irregularities their causes and corrective action that can be taken to cure the problem 3 3 1 Non Cyclic Noise Fluid Path Problems AU AU Time Time Unstabilized System Detector Flow Cell Leak AU TE ean Time Air Bubble in Flow Cell Possible Cause System Not Equilibrated Contaminated Mobile Phase Column Contamination Guard In Line Filter Contamination Air Bubble Trapped in Detector Flow Cell Electrochemical Detectors Only Air Bubble in Reference Electrode Air Bubbles in the Flow Path Corrective Action Allow the column detect
41. e should be taken when choosing a buffer and additive mixture to ensure that a solution of the two does not produce a solid salt which could cause System contamination Buffers should always be flushed from the analytical column and instrument after use to avoid salts being deposited on delicate frits etc Note There are LC MS instruments available for example the Finnigan Surveyor MSQ LC MS which incorporate a self cleaning mechanism to reduce the build up of inorganic buffers etc during routine use Care should still be taken not to purposefully over contaminate the instrument source as this will lead to operating difficulties 5 5 Chromophore Detection Wavelengths Chromophores are light absorbing groups Their behaviour is used to allow the detection of analytes They have one or more detection wavelengths each of which has a molar adsorbtivity associated with it The information contained in the following table is intended as a guide to common chromophores It is not an exhaustive list Chromophore Amax nm max L m cm Acetylide C C 175 180 6000 Aldehyde CHO 210 Strong 280 300 11 18 Amine NH 195 2800 Azidin gt C N 190 5000 Azo N N 285 400 3 25 Benzene 184 46 700 202 6 900 255 170 Carboxyl COOH 200 210 50 70 Ester COOR 205 50 Ether 0 185 1000 Ethylene C C 190 8000 Ketone gt C 0 195 1000 270 285 18 30 Napthalene 220 112 000 275 175 312 5 600 Nitrate ONO 270 12 XC C
42. e that a gradient program is not being used in place of an isocratic program and vice versa Replace the column with one where the performance is known Perform a series of standard injections and compare the peak retention times If they were reproducible then it would indicate that your original column is contaminated It can be cleaned using the procedures listed in section 5 6 of this guide If the erratic peak times continue the problem could be due to solvent immiscibility contaminated solvent or contaminated guards inline filters If guards are used it is advisable to remove them prior to the calculation of column efficiency Replace them with new units once the test is complete The most common cause of peak retention time drift in one direction is poorly prepared or mixed solvents or a system leak If you are confident that the solvents were prepared cor rectly then it is very important that you determine whether they are being mixed correctly mixing cell problems Where solvents are mixed manually prior to pumping ensure that the solvent flow rate is correct and constant Possible Cause Ambient Temperature Changes Flow Rate Changes Insufficient Equilibration Column Contamination Column Degradation Solvent Preparation Solvent Delivery System Blockage System Leaks 3 4 2 Continually Increasing or Decreasing Retention Times Corrective Action Refer to the previous table Ensure th
43. elp decrease retention selectivity and resolution 4 8 Sample Preparation Sample preparation is about more than just the dissolution of a solid in a liquid Samples may require other techniques such as filtration extraction or derivitization as well as accurate weighing and or dissolution Samples require filtration if they contain suspended solids This can be performed on line using a pre column filter or as the sample is introduced to the vial Important If using a membrane filter make sure that it is compatible with all the solvent used Samples that require extraction usually contain the analyte of interest at low levels The most common forms of extraction are liquid liquid solid liquid and solid phase extraction The latter of these three is perhaps the quickest and easiest to perform The question most often asked is when do perform an extraction The simple answer is i whenever the matrix that is the substance that your analyte is contained in is liable to contaminate your HPLC system or block it with particulate matter or ii whenever the analyte is at such low levels that pre concentration is required Solid phase extraction is not difficult to perform and requires very little specialist equipment Sample derivatization can occur before or after the column It can also be performed manually or automatically It is generally required for detection purposes for example the UV detection of analyt
44. ency The guard may also be the source of contamination so it is worth changing the guard at the same time as the column Replace contaminated or degraded guards and check the system perform ance It should improve If there is no improvement check for column contamination degradation as previously discussed The problem may be due to leaking or malfunctioning valves or a damaged or blocked needle Refer to the injector maintenance manual Check that the detector time constant setting is correct Change if necessary Normal Chromatography Abnormal Chromatography Possible Cause Injection Problem Detector Setting Error Recorder Input Error Corrective Action Ensure that the volume of sample injected is correct and that the sample is of the correct strength Dilute the sample or perform a smaller injection Check the wavelength zero and sensitivity settings Adjust if necessary Adjust the recorder input voltage QUICK TIP Tailing peaks are typically caused by column degradation or inlet contamination Carefully maintained columns and guards will considerably reduce the incidence of tailing peaks QUICK TIP Flat topped peaks are most often caused by either large injection volumes of dilute sample or by small injection volumes of strong sample solution 25 Negative peaks are most often caused by differences in refractive index between the s
45. ent mix that contains a minimum of 20 organic in water 4 3 Changing Solvents 4 3 1 Buffered Phase to Wash or Storage Phase Ensure that the buffer is soluble in the proposed wash or storage phase If it is not first flush the system with a solvent mix that is highly aqueous to remove the buffer from the system and column then change to the proposed wash or storage solvent mix 4 3 2 Normal to Reversed Phase and Vice Versa There are few occasions where these solvent changes will be necessary Such occasions include columns that can be used with both solvent types for example Hypercarb columns or systems where both normal and reversed phase analyses are performed Where possible systems should be dedicated to normal or reversed phase chromatography To convert a normal phase system column to a reversed phase system column flush with a solvent that is miscible with both the current normal phase solvents and ideally the proposed reversed phase solvents If the final reversed phase solvents include a buffer then it is advisable to move from the 100 methanol flush to a 50 aqueous methanol flush For example Normal Phase Hexane Ethyl Acetate Flush IPA then Methanol Finally 50 50 Methanol Water Reversed Phase Buffered Aqueous Methanol Important There are few columns that can be used in both normal and reversed phase Check that your column has been engineered to be compatible with both phase types before you attempt any solvent chang
46. erature Change the regularity of the on off frequency to avoid baseline noise Stabilize the air temperature around the instrument and allow the system to return to equilibrium If this is not possible relocate the instrument to a laboratory position where the detector is thermally stable and or avoid placing the instrument in direct sunlight Re Re Re Re Re Re Re er to the previous table er to er to er to er to er to er to Ambient Temperature Fluctuations in this table he previous table he previous table he previous table he previous table he previous table Contact your instrument maintenance provider The most common cause of cyclic baseline noise is the detector Usually if the detector is allowed insufficient time to equilibrate before an injection is performed then the resultant chromatogram will contain spurious peaks and there will also be some evidence of baseline drift The most common cause of peak retention time drift is an un equilibrated system The detector and fluid system must be stable prior to starting an analysis Temperature changes during the analysis are another major cause of peak drift If your analytical column is subject to fluctuations in temperature then we recommend that the column is housed in a thermally controlled environment such as a column oven jacket ete Finally where possible we recom
47. eries of commonly used HPLC solvents and their most pertinent physical properties including viscosity and miscibility number The miscibility numbers can be used to predict the miscibility of solvents If the smaller miscibility number is subtracted from the larger and the difference is 15 units or less then the two liquids are soluble in all proportions at 15 C If the smaller miscibility number is subtracted from the larger and the difference is 16 units then the two liquids have a critical solution temperature between 25 and 75 C with 50 C as the optimum temperature If the smaller miscibility number is subtracted from the larger and the difference is 17 or greater then the two liquids are immiscible or their critical temperature is greater than 75 C There is also a quick reference miscibility table in Appendix B of this guide Solvent Polarity Viscosity cp Boiling Point Miscibility Refractive UV Cut Off Index at 20 C C at 1 atm Number Index nm ActicAcd 62 12 4179 14 1372 230 Acetone 84 03 83 1 amp 9 139 Gs Acetonitrile 82 O97 816 WT 134 1 Cyclohexane 0 0 0 98 80 7 28 427 200 Ethyl Acetate 43 Off 1 1 137 26 Value refers to a 1 solution in water Solvents that have a double miscibility number are immiscible with other solvents at extremes of the lipophilicity scale The lower of the two numbers relates to solvents with high lipophilicity and the second to solvents with
48. es To convert a reversed phase system column to a normal phase system column follow a similar path to the one listed previously but in reverse for example Reversed Phase Buffered Aqueous Methanol Flush 50 50 Methanol Water Methanol then IPA Normal Phase Hexane Ethyl Acetate Refer to section 5 3 for solvent miscibilities and properties 33 4 3 3 General Before attempting any solvent change ensure that the solvent already in the system and column is compatible with the new solvent If the miscibility or physical properties of the two solvents are unknown then it is better to mix the solvents in a beaker to see the reaction than to go ahead and pump the second solvent into the first on the HPLC instrument Mixing problems are easier to rectify before the HPLC 4 4 System Plumbing and Fittings The purpose of a well plumbed HPLC system is to minimize dead volume between it s components and to eliminate leaks System tubing errors show themselves in many ways for example as band broadening baseline noise etc Detection of incorrect diameter tubing is often very difficult once it is in situ For this reason we recommend that all plumbing changes be recorded in the instrument maintenance log It is easier then to pinpoint the most recent changes An example of an instrument maintenance log is shown in Appendix C of this guide The internal diameter of tubing used in a HPLC system varies with the position in the instrument
49. es without chromophores There are many different derivitization techniques and care should be taken to choose one that is suitable for your application and that does not produce side products that will cause chromatographic problems later in the analysis Important Sample solvents should match the proposed mobile phase as closely as possible This will avoid baseline errors and spurious peaks seen as the injection solvent passes through the detector 5 Reference Data 5 1 Chromatographic Performance Tests 5 1 1 Resolution R Resolution is defined as the distance between two adjacent peak apexes divided by the average base width of both peaks It is represented by the equation T T R Q5 W Wj Where T and T are measured in seconds and are the peak apex retention times and W and W are the baseline widths of the peaks also measured in seconds Resolution is dependant on three other variables the column efficiency N the capacity factor k and the selectivity a Decreasing N decreases the resolution because peak width increases Increasing N increases resolution because peak width decreases Decreasing k sharpens the peaks but decreases resolution Increasing k broadens the peaks but improves resolution Increasing a increases resolution One peak moves relative to the other Likewise decreasing ot decreases resolution Where T Peak retentin time W Peak width at x height A Distance from apex to peak en
50. final action after cutting the tubing and before connecting it to the HPLC flush it with solvent to remove any filed material dust or other debris that could be in the tubing bore The tubing is now ready to have fittings attached to it C Metal tubing cutter Metal tubing cutter Polymeric tubing cutter 35 4 4 2 Fittings Fittings are available in stainless steel and a range of polymeric compounds In general you should use stainless steel fittings with stainless steel tubing PEEK fittings with PEEK tubing etc We recommend that compression fittings and ferrules from different manufacturers are never interchanged The dimensions of the fitting and ferrule vary between suppliers so mixing them often leads to leaks stripped threads and damage to female ports such as column inlet and outlets detector inlets etc The amount of tubing that extends past the ferrule also varies with manufacturer If the tubing does not seat properly within the female fitting then leaks and increases in dead volume will occur Ferrule does not seat Tubing is not seated caus ing unswept dead volume Tubing and ferrule seated correctly Laboratories that contain instruments from more than one manufacturer often find it useful to purchase universal fittings These fittings are compatible with all instruments Note Care should always be taken when tightening fittings Over tightening can cause damage to the fitt
51. h 120 mL dibutylether 3 Flush at 1 mL min with 50 mL acetone 4 Flush with aqueous mobile phase until equilibrated Normal Phase Regeneration Suitable for applications running predominantly in normal phase mobile phases 1 Flush at 1 mL min with 50 mL dichloromethane 2 Flush at 1 mL min with 50 mL methanol 3 Flush at 1 mL min with 50 mL water 4 Flush at 1 mL min with 50 mL 0 1M hydrochloric acid 5 Flush at 1 mL min with 50 mL water 6 Flush at 1 mL min with 50 mL methanol 7 Flush at 1 mL min with 50 mL dichloromethane 8 Flush with mobile phase until equilibrated Author A Karlsson Uppsala Sweden Removal of Trifluoroacetic Acid Suitable for applications running mobile phases containing trifluoroacetic acid 1 Flush the column with acetonitrile that has been heated to 75 C The column should also be maintained at this temperature 53 5 6 8 Polymeric Media with Metallic Counter lons There are three types of regeneration available for polymeric columns with metal counter ion Details of each procedure are listed in the following table Column Type Metal Contamination Organic Contamination Column Cleaning Hydrogen Counter lon Pump in reverse flow mode at Pump in reverse flow mode Pump in reverse flow mode 0 1 mL min with 0 1M H550 at 0 1 mL min with 20 80 at 0 1 mL min with 20 80 25 C for 4 to 16 hrs ACN H 0 25 C for 4 hrs ACN 0 01N H SO 65 C for 4 hrs Calcium Counter lon Pump
52. ices Ensure that all output signal switches are in the correct position Prepare new solvents prime all lines and the pump and allow the system to reach equilibrium Make sure that the sample injected is the correct one that it is of the correct strength and that it has not degraded Replace where possible If sample preparation techniques involved an extraction or similar ensure that the correct sample and solvents were used Where possible repeat the sample preparation and check that all reagents extraction equipment are correct and within their shelf lives Note Precipitation of the sample because of incompatibility with solvents will also result in no peaks being detected There is often a rise in system backpressure accompanying this problem U Normal Chromatography o Abnormal Chromatography often due to either the wrong sample being injected the detector not being switched on or a blockage between the injector and detector lines The next most common reason for a lack of peaks is that some part of the sample or mobile phase preparation has been performed incorrectly so it is always worth revisiting to check that the correct buffer has been used the sample solvent pH is correct etc Lack of chromatogram peaks is Smaller than expected peaks are often due to either the wrong sample being injected an incorrect sample volume being injected or a block
53. ing threads ferrules etc causing the unit to leak or in worse case scenarios causing the fitting to break off in the housing The use of fingertight fittings is recommended wherever possible These require no additional tools to form a leak proof seal Depth of Tubing Past Swaged Ferrule Fitting Length Excluding Ferrule amp Head Waters 3 3 mm Waters 8 1 mm Swagelok 2 3 mm Swagelok 5 7 mm Parker 2 3 mm Parker 5 3 mm Uptight 2 3 mm Uptight 6 4 mm Valco 2 0 mm Valco 7 6mm Rheodyne 4 3 mm Rheodyne 5 6 mm 4 4 3 SLIPFREE Fittings SLIPFREE fittings from Thermo ensure a void free connection on any HPLC system They are compatible with all end fitting depths and are fingertight to 10 000 psi They can be used between the injector and column between a guard and column or between the column and detector The SLIPFREE ferrule is firmly seated before the tubing depth beyond the ferrule is adjusted This gives the best fit possible as shown in the following images End fitting from a short seat column Tightening the screw thread nut creates a void until the locking nut is also tightened Once the locking nut is tightened the void is eliminated and the end fitting is compatible with a long seat column The locking nut allows the SLIPFREE tubing to extend into the fitting to ensure a zero dead volume seal in all column fittings For further information on these produc
54. ington PRISM StableBond Agilent Technologies SLIPFREE Supelcosil Sigma Aldrich UNIGUARD Swagelok Swagelok Company Symmetry The Waters Corporation SymmetryShield The Waters Corporatio SynChropak Eichrom Technologies Inc Teflon E l Dupont de Nemours and Co Tefzel E l Dupont de Nemours and Co TurboSEQUEST University of Washington Uptight Leco Corp Valco VICI Valco Instruments Co Inc Vydac W R Grace and Co Waters The Waters Corporation Waters Spherisorb The Waters Corporation YMC The Waters Corporation XTerra The Waters Corporation ZirChrom ZirChrom Separations Inc Zorbax Agilent Technologies AquaSil Siliconizing Fluid for treating glass surfaces is sold by Pierce Chemical Co Rockford IL State of the art instruments are only the beginning with Thermo Electron Comprehensive service and support programs are offered on our products worldwide by a network of factory trained and highly qualified scientists and engineers Our experts help you choose the right instruments for your lab then keep the instruments performing to specification Contact us today for more information on how our specialized sales and service engineers can help you meet your laboratory needs In addition to the Thermo Electron offices listed below we have an extensive network of authorized global distributors for Thermo Electron HPLC columns For the list of international distributors
55. is not being injected that the sample vial contains enough solvent to perform multiple injections from it and that there are no leaks in the system Ensure that the sample loop is the correct size and that the injection system is undamaged and clean Between injections ensure that the injector purge is adequate to eliminate carry over from previous injections Perform one injection of a standard If all the peaks are present and correct the sample has been contaminated or has degraded Re prepare the sample and re inject Refer to the troubleshooting tables in section 3 4 of this guide Refer to the detector operator s manual for instrument specific information on troubleshooting and corrective action Ensure that the correct values of peak lift off touchdown threshold etc are entered into the data handling device Make any necessary changes Note Many regulatory bodies insist that all chromatograms in a run are integrated using the same integration parameters For this reason it is considered best practice to set the integration parameters using a system suitability test mix that contains peaks at or just above the limit of detection and larger 4 Good Laboratory Practice For HPLC 4 1 Preparation of Solvents Correct solvent preparation is very important It can save vast amounts of time spent troubleshooting spurious peaks base line noise etc 4 1 1 Quality All reagents and solvents should be of the highest qu
56. ite lifetime Eventually they will have to be replaced with new ones The ferrule of the worn fitting will be swaged to the tubing i e it will be irremovable stainless steel or will be removable but will leave an indentation around the tubing PEEK This section of tubing cannot be used again as it would not seal properly and would leak To change a fitting follow the guideline below Compression Fitting 1 Turn off all pumps solvent should not be flowing through the system 2 Remove the fitting from it s housing 3 Cut the tubing between the ferrule and fitting If this is not possible cut the tubing before the fitting 4 Ensure the tubing s cut end is flat and burr free Inspect the old fitting if it is worn replace it If it is not worn it can be re used with a new compatible ferrule Note Leaking fittings are often caused by damaged mis shapen ferrules so it is not necessary to replace the whole fitting if only a part of it is damaged 5 Assemble the new fitting and ferrule on the tubing and tighten into it s housing to swage the ferrule onto the tubing If the fitting still leaks after this replace the tubing Fingertight Fitting 1 Turn off all pumps solvent should not be flowing through the system 2 Remove the fitting from it s housing 3 Slip the fitting and ferrule if it has a separate one from the tubing 4 Inspect the tubing for defects Remove the end of the tubing if necessary 5 Place
57. le 5 pm in diameter L43 Pentafluorophenyl groups chemically bonded to silica particles 5 10 um in diameter Fluophase PFP 5 yum spherical yes use for halogenated compounds and shape selectivity L44 A multifunctional support which consists of a high purity 60 spherical silica substrate that has been bonded with a cationic exchanger sulfonic acid functionality in addition to a conventional reversed phase C8 funtionality L45 Beta cyclodextrin bonded to porous silica particles 5 10 pm in diameter L46 Polystyrene divinylbenzene substrate agglomerated with quaternary amine functionalized latex beads 10 um in diameter L47 High capacity anion exchange microporous substrate fully functionalized with trimethylamine groups 8 um in diameter L48 Sulphonated cross linked polystyrene with an outer layer of submicron porous anion exchange microbeads 15 um in diameter L49 A reversed phase packing made by coating a thin layer of polybutadiene onto spherical porous zirconia particles 3 10 um in diameter L50 Multifunction resin with reversed phase retention and strong anion exchange functionalities The resin consists of ethylvinylben zene 55 cross linked with divinylbenzene copolymer 3 15 pm in diameter and a surface area not less than 350m2 g Substrate is coated with quaternary ammonium functionalized latex particles consisting of styrene cross linked with divinylbenzene L51 Amylose tris 3 5 dimethylphenylcarbamate
58. low lipophilicity Solvents with double miscibility numbers can in some circumstances be immiscible with each other Important molecular interaction hydrogen bonding for example between liquids can change the expected level of miscibility The following tables list a series of commonly used HPLC buffers their alternative name where applicable and their pK values at 20 C 5 4 Buffer Properties Buffer transparency is a variable that should be measured prior to buffer use as it will vary with salt concentration Buffer Buffer Range pK at 200C ACES N 2 Acetamido 2 aminoethanesulfonic Acid 6 4 7 4 6 9 Acetamidoglycine N 2 Acetamido Glycine 7 12 Acetic Acid Ammonium K amp Na Acetate 3 8 58 48 ADA N 2 Acetamido iminodiacetic Acid 64 74 6 6 Mono amp Di Ammonium K amp Na Carbonate 54 74 6 4 93 113 10 3 Ammonium Hydroxide Chloride Ammonia 8 2 10 2 9 2 BES N N Bis 2 hydroxyethyl 2 aminoethane sulfonic Acid 6 6 7 6 7 15 Bicine N N Bis hydroxyethyl glycine 7 8 8 8 8 35 BIS TRIS Propane 1 3 Bis tris hydroxymethyl methylamino propane 5 8 7 8 6 8 Borate 82 10 2 9 24 CAPS 9 7 11 1 10 40 CHES 9 0 10 1 9 55 Cholamine Chloride 2 Aminoethyl trimethylammonium 74 Chloride Hydrochloride Citric Acid Tri potassium citrate 2 1 6 4 3 1 54 Diethylamine Hydrochloride Diethylamine 9 5 11 5 10 5 Formic Acid Ammonium K amp Na Formate 2 8 4 8 3 8 Glycinomide Glycinamide Hydrochloride 8 2
59. m the main supply before the removal of their covers etc Wear eye protection when troubleshooting the detector with the cover removed as ultraviolet light is emitted during UV and fluorescence detector operation and this will damage the eye s cornea irreversibly There are many areas in a HPLC instrument that can give rise to system and chromatographic problems This guide will deal with each one in the following sections 3 1 Visual Inspection 3 2 Pressure 3 3 Baseline Irregularities 3 4 Changes in Chromatography 3 5 Qualitative Results 3 6 Quantitative Results Chromatography Data Systems CDS and in depth hardware investigations will not be included in this guide as they are outside its scope We recommend troubleshooting these systems with your supplier 3 1 Visual Inspection When a problem occurs it is advisable to perform a quick visual check of the instrument and column This will pick up leaks loose or disconnected tubing changes in instrument settings etc These problems are easy to rectify and will save time 3 2 Pressure System pressure is affected by a number of variables including the viscosity of the solvent used column variables flow rate and temperature It is important to have a reference point when comparing high or low pressures to the norm This reference point should be the pressure generated in the system when everything is functioning correctly It is a good idea to note the system pressure under n
60. mend the pre mixing of all solvents used in isocratic methods 3 4 Changes in Chromatography The most common changes in chromatographic response are related to the shape and separation of peaks their elution times and changes in established performance There are two stages to troubleshooting unacceptable chromatography First the evaluation of what you have chromatographically speaking and secondly isolation of the source of the problem The evaluation stage is best performed on a standard rather than sample injection The nature and prior chromatographic performance of the standard should be recorded each time it is injected This will provide historical data for any comparisons that you need to perform A typical record sheet for recording standard data is shown in Appendix E The following sections will list each of the most common causes of change in chromato graphic response and corrective action that can be made to return your chromatography to its previous state 3 4 1 Retention Time Changes from Injection to Injection Possible Cause Corrective Action System Not Equilibrated Allow the column detector etc sufficient time to stabilize If performing gradient analysis allow sufficient time between analyses for the system to re equilibrate The first time ion pair reagents are used with a column allow sufficient time and volume of solvent to adequately equilibrate the column Insufficient Equilibration A suitable perio
61. ment manufacturer The typical solvent flow rate through a column is very dependent on it s internal diameter and the particle size of the column packing material The flow rates shown in the following table are intended as a guide only and allowing for experimental differences it is possible to use a range of flow rates around those quoted Internal Diameter Media Particle Size Typical Flow Rate mm um mL min 1 0 5 0 1 2 0 2 1 5 0 2 3 0 5 0 5 4 0 4 6 3 0 5 5 1 0 10 2 0 10 0 5 5 0 21 2 10 21 0 5 2 2 Scaling Up or Scaling Down Flow rate and column load scaling are only required when changing column internal diameters Scaling up is performed when moving from a small to a large diameter column for example analytical to preparative chromatography Scaling down is performed when moving from a large to a small diameter column for example analytical to capillary chomatography The scaling allows peak retention times to remain relatively constant between different dimension columns Assuming column length is a constant the scale factor can be calculated using the following formula Column A internal diameter Scale Factor i Column B internal diameter As an example the calculated scale factors based on a 4 6 mm internal diameter column are New Column ID mm Flow Rate Scale Factor 1 0 x 0 05 2 1 x 0 2 3 0 x 0 4 40 x0 8 10 0 x47 212 x212 47 5 3 Solvent Properties The following tables list a s
62. ming gradient analysis allow sufficient time between analyses for the system to re equilibrate The first time ion pair reagents are used with a column allow sufficient time and volume of solvent to adequately equilibrate the column Set the chart speed to a faster rate Contamination Clean the column following the guidelines in section 5 6 of this guide If the problem remains after cleaning replace the column Column degradation Replace the column with one where the performance is known Perform a series of standard injections and compare the peak retention times If they were reproducible then it would indicate that your original column is contaminated It can be cleaned using the procedures listed in section 5 6 of this guide If the erratic peak times continue the problem could be due to solvent immiscibility contaminated solvent or contaminated guards inline filters If guards are used it is advisable to remove them prior to the calculation of column efficiency The guard may also be the source of contamination so it is worth changing the guard at the same time as the column Check that the column in use is of the correct dimension and is packed with the correct media for the application Replace contaminated or degraded guards and check the system perform ance It should improve If there is no improvement check for column contamination degradation as previously discussed All Peaks Doubling Normal Ch
63. mixture with specific concentrations of each impurity Most real life samples contain impurities at very low levels so the retention time of their peaks will be slightly different to those generated by the standard mix Identification windows should be set widely enough to take into account this time variation with respect to concentration Extra Peaks Possible Cause Contaminated Solvents Mobile Phase Gradient Methods Contaminated Degraded Sample Fluid Path Noise Sample Loop Flush Inadequate Corrective Action Discard all old solvents Prepare fresh buffers etc and place in clean dry reservoirs Purge all lines and the pump with the new solvents Allow the system to come to equilibrium and perform a standard injection The spurious peaks should not be present Ghost peaks can be caused by changes in mobile phase composition during gradient methods An example of such a peak is shown in the chromatograms that follow this table Perform one injection of a standard If all the peaks are present and correct the sample has been contaminated or has degraded Re prepare the sample and re inject Refer to section 3 3 of this guide Ensure that the sample loop is thoroughly flushed with solvent between injections to avoid carry over Contaminated Injector Flush the injector with solvent If necessary replace all perishable parts for example seals and filters Contaminated Column Clean the column follo
64. mn Incorrect Ensure that the correct media and dimension of column is used Also check that the correct media particle size is used as this will have an impact on the resolution of closely eluting peaks a 3 um media will provide higher column efficiency than a 5 um media Solvent Flow Program Inconsistencies Verify that the analysis uses the correct proportions of solvents and that the correct gradient or isocratic path is being followed Check that the solvent flow rate is accurate from all lines and that the correct flow rate is entered into the pump software If a gradient is used check that the time between analyses is sufficient to allow the system to re equilibrate No Peaks Found Refer to section 3 4 4 of this guide Single or multiple missing peaks are usually due to the wrong sample being injected or the sample degrading Equally likely though is a loss of resolution due to column solvent inconsistencies 27 Quick TiP Extra peaks in chromatograms are more often than not due to contamination or degradation of the sample mobile phase or column To check if the extra peak s is are in the sample alone perform a blank injection of sample solvent The peak s should be absent QUICK TIP Peak mis identification occurs most often in degradation samples or those in which related substance levels are being measured This is because the software is calibrated using a standard
65. mooth jawed pliers one above the score and one below it gently bend the tubing back and forth until it snaps Excessive bending should be avoided as this will damage the tubing and not give Y a clean break void caused by tube end angle 4 There will be one or two burrs present on the cut surface These can be carefully filed away to give a smooth flat cut surface Care should be taken not to allow any filed material to block the tubing bore PEEK or Polymeric Tubing The simplest way to cut polymeric tubing is using a blade for example a razor blade or craft knife The polymeric tubing cutters that are supplied by many manufacturers are essentially a razor blade in a safety housing The advantage of using one of these cutters is that they hold the tubing at 90 to the blade so a clean flat cut is assured Polymeric tubing can be effectively cut using the procedure outlined below 1 Estimate the length required Remember to allow extra length if the tubing is to go around corners as sharp bends in the tubing will distort the inside bore and hence the solvent flow through it 2 Use a sharp blade or specialist cutter to cut the tubing Do not use a sawing action This will give an uneven cut surface Make the cut in a single action 3 Inspect the cut surface for burrs These can be carefully filed away to give a smooth flat cut surface Care should be taken not to allow any filed material to block the tubing bore As a
66. ng or extra peaks and b peak mis identification The following tables will assist in tracing errors in qualitative methods Missing Peaks Possible Cause Corrective Action Incorrect Solvents Used Ensure that the solvents used are correct for the method sample Prepare fresh solvent if necessary Note if the problem occurs with an increase in system pressure then this would indicate sample precipitation within the LC system Follow the flow chart in section 3 2 1 to determine where the increase in pressure originates Sample Degradation Perform one injection of a standard If all the peaks are present and correct the sample has degraded Re prepare the sample and re inject Resolution Lost Replace the column with one where the performance is known and re inject the sample If the missing peak s re appear then the efficiency of your original column may be lower than that of the replacement column Check the efficiency of the original column using a test mix and conditions similar to those used by the column manufacturer to test efficiency when the column was new For columns that have been in use for an extended period of time perform a column clean up as per section 5 6 of this guide Repeat the efficiency test to determine whether this has improved column performance You may wish to contact your column supplier if the low efficiency column is relatively new to discuss whether it was a handling error that caused the problem Colu
67. nic and cationic water soluble polymers A polymethacrylate resin base cross linked with polyhydroxylated ether surface contained some residual carboxyl groups was found suitable L26 Butyl silane C4 chemically bonded to totally porous silica particles 5 10 yum in diameter HyPURITY C4 5 um spherica yes use for acids bases neutrals or chelators BioBasic 4 5 um spherica yes use for peptides proteins and other biomolecules BetaBasic 4 3 and 5 um spherica yes 150 appropriate for most applications Hypersil C4 3 5 and 10 um spherica use for acids and neutrals L27 Porous silica particles 30 50 yim in diameter HyperPrep Silica 30 um spherica yes Phase Particle Size Particle Shape End Capped Comments L28 A multifunctional support which consists of a high purity 100 spherical silica substrate that has been bonded with anionic exchanger amine functionality in addition to a conventional reversed phase C8 functionality L29 Gamma alumina reversed phase low carbon percentage by weight alumina based polybutadiene spherical particles 5 um in diameter with a pore diameter of 80A L30 Ethyl silane chemically bonded to totally porous silica particle 3 10 um in diameter L31 A strong anion exchange resin quaternary amine bonded on latex particles attached to a core of 8 5 pm macroporous particles having a pore size of 2000A and consisting of ethylvinylbenzene cross linked with 55 divinylbenzene L32 A chiral lig
68. nol 3 Flush with chloroform 5 6 4 Cation Exchange Media 1 Flush with HPLC grade water inject 4 aliquots of 200 uL DMSO during this flush 2 Flush with tetrahydrofuran 5 6 5 Protein Size Exclusion Media There are two wash regeneration procedures associated with the removal of contaminants from protein size exclusion media Weakly retained proteins 1 Flush with 30 mL 0 1 M pH3 0 phosphate buffer Strongly retained proteins 1 Flush for 60 minutes using a 100 water to 100 acetonitrile gradient 5 6 7 Porous Graphitic Carbon There are four wash or regeneration procedures associated with porous graphitic carbon The one s used will depend on the analytes and solvents that have been used with the column Acid Base Regeneration Suitable for ionized species analyzed in strongly aqueous mobile phases 1 Invert the column 2 Flush at 1 mL min with 50 mL tetrahydrofuran water 1 1 containing 0 1 trifluoroacetic acid 3 Flush at 1 mL min with 50 mL tetrahydrofuran water 1 1 containing 0 1 triethylamine or sodium hydroxide 4 Flush at 1 mL min with 50 mL tetrahydrofuran water 1 1 containing 0 1 trifluoroacetic acid 5 Flush with methanol water 95 5 to re equilibrate 6 Re invert the column Author R Plumb Glaxo UK Strong Organic Regeneration Suitable for applications involving polar and or ionized species analyzed in aqueous mobile phases 1 Flush at 1 mL min with 50 mL acetone 2 Flush at 1 mL min wit
69. ns the same level of noise even after changing the column then it indicates that the noise is due to another cause such as solvent miscibility contaminated mobile phase or contaminated guards in line filters Guard cartridges and in line filters are designed to be disposable We do not recommend attempting to clean up these items as the costs involved in time and materials out weights the cost of part replacement To remove the air bubble either purge the detector flow cell or apply a slight pressure to the detector waste outlet The air bubbles usually originate from poorly degassed mobile phase so once the bubble is removed it is advisable to thoroughly degas the phases again To stop air bubbles forming in the flow cell attach a 30 to 90cm length of 0 23 mm 1D 1 58 mm OD tubing to the detector water outlet The tubing acts as a flow restrictor increasing backpressure in the cell When adding the tubing please be aware of the backpressure limits of the flow cell Note 90 cm of tubing will produce 30 to 50 psi of backpressure at 1 mL min Remove the reference electrode from the instrument and gently shake it to dislodge the air bubble Prime the pump once again and ensure that all solvents are thoroughly degassed QUICK TIP The most common cause of non cyclic baseline noise related problems is air in the system To overcome this all solvents should be thoroughly degassed prior to use all lines should be pu
70. odium metabisulphite These stabilizing agents often affect the optical and chromatographic behaviour of buffer solutions so it is often worth buying reagents that contain no stabilizer Containers of solid reagent are easily contaminated by repeated use For this reason we recommend that regents be purchased in low container weights 4 1 3 Filtration Ideally all HPLC solvents should be filtered through a 0 45 um filter before use This removes any particulate matter that may cause blockages After filtration the solvents should be stored in a covered reservoir to prevent contamination with dust etc Filtering HPLC solvents will benefit both your chromatography and the wear and tear of the HPLC system Pump plungers seals and check valves will perform better and lifetimes will be maximized 4 1 4 Degassing Before the freshly prepared mobile phase is pumped around the HPLC system it should be thor oughly degassed to remove all dissolved gasses Dissolved gas can be removed from solution by Bubbling with helium Sonication Vacuum filtration If the mobile phase is not degassed air bubbles can form in the high pressure system resulting in problems with system instability spurious baseline peaks etc 80 80 60 2 604 2 2 5 s z 404 1 404 2 5 204 204 2a ra g 04 04 A 20 20 7 BER EES SEP BS SE SSL E SEE LL SS T T TTT T T T T T 0123 45 678 9 o 123 45 6 7
71. ograms using the same chromatographic conditions Refer to the table in section 3 4 1 Refer to the table in section 3 4 1 Check the maintenance schedule for the pump fluid system to find out whether there have been any changes since the system was last used If a change has been made re calculate the new gradient delay volume Refer to the pump operation manual for details of this calculation or contact the instrument manufacturer The most common cause of peak retention time drift to a new constant value direction is poorly prepared or mixed solvents If you are confident hat the solvents were prepared correctly then it is very important that you determine whether they are being mixed correctly mixing cell problems Where solvents are mixed manually prior to pumping ensure that the solvent flow rate is correct and constant The last most common cause of retention time change is a leak in the system or build up of contaminants 3 4 4 Abnormal Peak Shape Abnormal peak shape encompasses a range of possible peak shape problems No peaks Fronting or tailing peaks Smaller than expected peaks Double peaks shouldering peaks Broad peaks early eluting analytes or all analytes Flat topped peaks Negative peaks If all the peaks in a chromatogram are affected then it suggests that the problem is related to either the system or the column If only early eluting peaks are affected then it suggests that
72. olvents volatile Yes Go to next question Go to next question If pressure changes follow the gradient then this may be normal depending on the individual solvent viscosities Go to next question Go to next question Go to next question Ensure that the operating temperature is suitable for the particular solvent used Degas thoroughly 8 Pump problem Contact your maintenance provider No Set the flow to zero Is the pressure stable Adjust the transducer Can the pressure be zeroed Defective transducer Refer to the pump maintenance manual Prime the pump again Go to next question Refer to the pump maintenance manual Bubble with an inert gas or place in an ultrasonic bath to remove dissolved gases Check the solvent miscibility using section 5 3 of this guide Go to next question The most common cause of fluctuating pressure is poorly primed lines with badly degassed solvents 3 3 Baseline Irregularities Baseline irregularities can be non cyclic erratic or cyclic follow a pattern They can originate from electrical interferences detector faults solvent impurities column contamination etc To isolate the source of a baseline irregularity it is important to determine whether the problem lies with the fluid path detector or electrical connections This can be achieved by following the simple steps below 1 Turn off the instrument pump fluid flow must be zero 2 Monitor th
73. on 5 6 of this guide Repeat the efficiency calculation If there is no increase in efficiency replace the degraded column with a new one You may wish to contact your column supplier if the degraded column is relatively new to discuss whether it was a handling error that caused the degradation Replace contaminated or degraded guards and check the system performance It should improve if there is no improvement check for column contamination degradation as previously discussed Tailing Peaks LoL ue aie Normal Chromatography Abnormal Chromatography Possible Cause Column Problems Guard Problems Injector Problem Detector Problem Flat Topped Peaks Corrective Action Column contamination Clean the column following the guidelines in section 5 6 of this guide If the problem remains after cleaning replace the column Column degradation Replace the column with one where the perform ance is known Perform a series of standard injections and compare the peak retention times If they were reproducible then it would indicate that your original column is contaminated It can be cleaned using the procedures listed in section 5 6 of this guide If the erratic peak times continue the problem could be due to solvent immiscibility contaminated solvent or contaminated guards inline filters If guards are used it is advisable to remove them prior to the calculation of column effici
74. onvenience 4 6 Column Protection 4 6 1 Guard Columns and Cartridges It is advisable to protect your analytical column from sample and system debris and contaminants to maintain the column performance and efficiency Guard columns or cartridges are one of the most cost effective and efficient ways of trapping these unwanted system components We recommend a 10 mm length guard for moderate to heavy contamination Note Guards are designed to be disposable so once contaminated they should be replaced rather than regenerated Dolan and Snyder recommend that the guard is replaced every 50 100 injections to ensure that there is no loss in column performance The performance of an analytical column should not be affected by adding a guard unit to the system As an example the following chromatograms of procainamides were generated both with and without a C18 guard unit As you can see there is no change in peak separation shape or analysis time Without a guard With UNIGUARD C18 Analytes Procainamides o 8 MIN 0 8 MIN The choice of guard often causes concern The simple rule of thumb is to choose a guard that is packed with the same particle size and type of material as the analytical column The guard and column diameters should be as close as possible to eliminate solvent flow and chromatographic discrepancies A reference table is provided on the left for your convenience Specialist guar
75. or any problem is required 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 Compare the performance obtained to the expected performance 4 List possible causes 5 Work through the possible causes in a step by step manner checking the outcome from any changes made 6 As a last resort get help from elsewhere for example your instrument supplier help desk or your local technical support department It is important to remember that once the problem is defined and possible corrective action is identified only one change at a time should be made after each change the whole system should be checked again to determine whether the problem still exists or whether the change corrected the problem All problems and corrective action should be documented in the instrument log An example instrument log is available in Appendix F for your convenience 3 Troubleshooting Isolation amp Corrective Action Before starting any troubleshooting whether it is related to instruments or columns it is essential that safe laboratory practices be observed The chemical and physical properties of any solvents used should be known and the Material Safety Data Sheet MSDS for these solvents should be readily available All electrically powered instruments should be powered down and unplugged fro
76. or etc sufficient time to stabilize If performing gradient analysis allow sufficient time between analyses for the system to re equilibrate The first time ion pair reagents are used with a column allow sufficient time and volume of solvent to adequately equilibrate the column Do not use mobile phase that is contaminated or thought to be contaminated Thoroughly wash the reservoir that contained the contaminated phase Ensure that no traces of detergent remain in the vessel as this will cause spurious peaks in the baseline Clean all solvent inlet filters in a sonic bath using 6N nitric acid followed by water then finally methanol Prepare fresh mobile phase and purge the solvent lines with this phase Close the purge valve and pump the new phase around the HPLC system to flush out any remaining contaminated phase Allow the system to equilibrate prior to use To determine whether the column is contaminated replace it with a new column or a column where the performance is known Flush the column with mobile phase and monitor the baseline A baseline free from the previous noise indicates that the original column was contaminated To clean the contaminated column refer to the guidelines in section 5 6 of this guide Please be aware that not all columns can be cleaned and not all contaminants can be removed from column beds In such circumstances it is prudent to replace the column with a new one immediately If the baseline contai
77. ormal operating conditions each day or each time your HPLC is used This will allow you to spot any pressure trends that otherwise might go unnoticed Pressure problems fall into one of three categories high low or fluctuating pressure They can occur suddenly or be a gradual process Sudden pressure rises tend to be due to particles from the sample blocked or damaged tubing or column packed bed collapse Gradual pressure rises can also be due to particles in the sample but they can also arise from particles generated in the instrument for example debris from vial septa or degrading seals Before releasing any high pressure build up in a system be aware that the solvent may form an aerosol or spray when loosening connections Eye protection should be worn and ideally the connection to be loosened should be positioned above an adsorbent material to soak up all released solvents The simplest way to troubleshoot pressure problems is using a systematic approach as highlighted in the following tables for high low and fluctuating pressure Note Subsequent tables will be displayed using a cause and effect layout with the most common fault entered first into the table 3 2 1 High Pressure High Pressure Reading Question 1 Has the ambient temperature changed 2 Is the flow rate correct 3 Is the eluent viscous 4 Is the pressure transducer operating correctly 5 Loosen detector waste outlet fitting Does the pressure return
78. rged with solvent and the pump should be thoroughly primed QUICK TIP Air bubbles can obscure the detector flow cell and cause baseline noise be aware that from time to time the cell may require cleaning and or removal of air bubbles The most common cause of problems related to electronic baseline noise is the detector Usually if the detector is allowed insufficient time to equilibrate before an injection is performed then the result ant chromatogram will contain spurious peaks and there will also be some evidence of baseline drift If the problem occurs after routine maintenance check that all the cables are securely seated in their sockets and that the correct cable is in the correct socket Also check that all settings have been returned to their positions prior to the routine maintenance 3 3 2 Non Cyclic Noise Detector Electronics Problems AU Time Defective Lamp AU M uc Time High Sensitivity Possible Cause Detector Not Stable Detector Lamp Malfunction Contaminated Detector Flow Cell Detector Electronic Problem Cables Radio Interference Gain Sensitivity Setting Too High Reference Electrode Leak Dirty Reference Electrode Contaminated Scratched Reference Electrode Corrective Action After turning the detector on allow it sufficient time to stabilize The baseline will be stable once the detector is s
79. romatography Possible Cause Blockage Partial Blockage Before the Column Column Voiding Guard Column Voiding Injection Disrupting Equilibrium L Abnormal Chromatography Corrective Action Check the guard in line filter column inlet and all associated tubing for blockages Replace any blocked tubing filters or guard units If the column inlet frit has been blocked gently back flush the column with a wash solvent at very low flow rate preferably over night for best effect Re invert the column and equilibrate with test solvent If this procedure has not removed the blockage please contact your column supplier for further technical advice Using a standard mixture solvent and analysis conditions as close to those used to generate your column s test certificate as possible calculate the columns efficiency N If N is markedly lower than the manufacturer s test certificate value try cleaning the column using the procedures in section 5 6 of this guide Repeat the efficiency calculation If there is no increase in efficiency replace the degraded column with a new one You may wish to contact your column supplier if the degraded column is relatively new to discuss whether it was a handling error that caused the degradation Remove the defective guard and replace with a new one Allow the system to reach equilibration and repeat the sample injection If the problem persists remove the guard and hol
80. stituted benzene 715 008a 16 20 4 B 32 k of Phenylheptane Reversed Phase Chemistries Thermo reversed phase materials are based on high purity silicas giving excellent chromatography of acids bases and neutral compounds Our premium columns include HyPURITY and BioBasic families in a wide range of chemistries an excellent choice for all of your chromatography needs Choose any of our families for method development LC MS and your most demanding separations All of our reversed phase materials offer superb reproducibility and reliable performance for routine and QC analyses Our experienced Technical Support staff are always able to help you select the appropriate column for your application Premium Columns Hypersil GOLD Columns The key to outstanding peak shape e Exceptional peak symmetry and resolution e Based on next generation ultra pure silica Outstanding pH stability e Ideal first choice for new method development HyPURITY Columns The choice for superior chromatography e Ultra pure highly stable silica for long column lifetime reliability and performance Excellent peak shapes for acids bases chelators and neutral compounds 190A pore size for small molecules peptides and protein digests e Family of phases for a variety of selectivities e Ideal for LC MS applications e Excellent alternative to other highly pure silica columns BioBasic Columns Better chromatography of biomolecules 300A pore size for
81. sufficiently tight to stop evaporation but not tight enough to create a vacuum when injections are performed Degraded Contaminated Sample Refer to the table detailing loss of precision Peak Integration Error Refer to the table detailing loss of precision Loss of accuracy is most often related to the sample 29 Loss of precision is most often caused by an injector error by a sample that is mixed poorly or a sample that is degrading Loss of Precision Possible Cause Injection Error External Standard Degraded Contaminated Sample Chromatographic Errors Detector Response Inaccuracies Peak Integration Error Corrective Action Manual Injection Using a fixed loop system load three times the loop volume before making the injection Using a partial fill loop Inject less than 50 of the sample loop volume Syringe and injection valve Ensure that the injection technique is as con stant as possible This method of performing injections is subject to human error in addition to instrumentation errors so you must take this into account when determining an acceptable level of precision for the method Check that the loop size and syringe are correct and uncontaminated Use a syringe where not less than 20 of the full volume is injected Finally ensure that the injection port is not leaking and that any switches open and close to their full extent Automatic Injection Check to make sure that air
82. sure cut off to a value below the operating pressure Go to next question Go to next question Remove the air check for loose connections Go to next question Go to next question Go to next question Check for pools of liquid buffer crystals and loose connections Clean up and stop leaks where necessary Go to next question Go to next question Take the auto injector out of prime mode refer to injector operation manual Go to next question 17 Pump problem Contact your maintenance provider No Replace fuse and re test Turn on the pump Go to next question Refill the reservoir and re test Go to next question In line filter blocked Clean as per the pump manual instructions Prime as per the pump instruction manual Go to next question Refer to pump head maintenance in the pump manual Set the correct flow rate Maintain column at a constant temperature Go to next question Use the correct solvent composition Close the purge valve Go to next question Check the actual versus theoretical flow for each solvent line Refer to the pump maintenance manual 3 2 3 Fluctuating Pressure Fluctuating Pressure Reading Question 1 Is the pressure transducer functioning correctly 2 Was the pump primed properly 3 Are you performing a gradient analysis 4 Are the pump heads functioning correctly 5 Are all the solvents degassed 6 Are all solvents miscible 7 Are the s
83. t solution Inject 5 uL of this diluted mix 4 Adjust the detector sensitivity until the peak height is approximately 75 of the full scale readout 5 Measure the peak width at 4 4 peak height 5 sigma column efficiency method 8 Convert the peak width to mL using the following conversion Band Spread uL Peak Width x 1 20 x 1 x 1000 Where Peak width is measured at 4 496 peak height and expressed in cm 1 20 represents the chart speed min cm 1 represents the flow rate mL min and 1000 represents the volume correction factor uL mL Important 100 mL 30 uL is a typical system band spreading value Larger values may indicate a problem in the detector injector tubing or fittings Ensure that your HPLC system does not have built in extra dead volume as this will also increase the band spreading value 7 For high values of band spreading troubleshoot your HPLC system then repeat the determina tion of band spreading If the value decreases to acceptable levels then the problem is resolved A partial decrease will require further investigation If the problem persists contact your instrument supplier for technical advice The table below shows details of the volume of solvent per unit length contained in tubing of varying volumes For ease of use both metric and imperial measurements are shown Tubing Diameter Tubing Volume Tubing Diameter Tubing Volume mm pL cm inch pL inch 0 12 0 127 0 005 0 323 0 17 0 2
84. tabilized Different detectors and conditions will require different stabilization times We recommend referring to your detector manual for guidance Check that the lamp energy and reference energy are within specification limits for normal detector operation refer to your detector maintenance manual for guidance If the lamp energy is below that recommended for normal detector operation replace the lamp Note Some models of detector allow the lamp energy level to be manipulated when the lamp nears the end of its useful lifetime Refer to your detector maintenance manual for details if applicable Clean the flow cell as per the maintenance instructions given in your detector manual Alternatively remove the analytical column and replace with a union Flush the system with water followed by methanol then water to remove any excess buffers ensure that water and methanol are compatible with the last solvent to pass through the flow cell The detector flow cell can also be cleaned with a 50 50 v v mixture of THF water then 100 THF if the system is used in normal phase Once again ensure that the solvents used in the cleaning procedure are compatible with that last used in the flow cell Contact your maintenance provider Check that all cables are securely seated in their respective terminals Ensure that all output switches have the correct setting are in the correct position All cables should be well maintained and gro
85. the new fitting on the tubing and tighten it into it s housing If the fitting still leaks after this replace the tubing SLIPFREE Fitting 1 Turn off all pumps solvent should not be flowing through the system 2 Remove the fitting from it s housing 3 Check the ferrule for damage this is the most likely cause of leaks The SLIPFREE ferrules do not swage onto the unit s tubing unlike other ferrules so they can easily be replaced 4 Replace the ferrule and reassemble the SLIPFREE connector into it s housing If the fitting still leaks replace the entire SLIPFREE unit Important There are occasions where the fitting or ferrule is not the cause of solvent leaks They can be generated by degeneration of the fitting housing Where persistent leaks occur even after fitting changes it is worth while inspecting the housing for damaged threads blockage etc 4 5 Column Selection For applications where there is no specified column type or in method development situations column selection is vitally important It can mean the difference between efficient and inefficient method development There are many variables to consider such as the analyte properties and available chromophores To make your selection choices easier refer to pages 61 and 62 or contact your Thermo representative Reference tables of hydrophobic character and wavelength selection for chromophores are included in Appendix A and section 5 5 of this guide for your c
86. there is no analyte carry over from previous injections especially from previous injections where gradient analyses are being performed Determine whether the broad peaks are due to strongly retained analytes from a previous injection by performing a single injection of sample and increasing the run time Important Insufficient equilibration time between injections where a gradient analysis is perform can also affect peak shape Check that all wait times between injections are sufficient to allow reproducible chromatography Broad early eluting peaks are most commonly associated with sample overload or incorrect system plumbing 21 All Peaks Broad Normal Chromatography Abnormal Chromatography Possible Cause for the Mobile Phase Ambient Temperature Change Column Quick TIP Broad peaks all are most often due to errors in instru mentation or column It is worthwhile investigating the column and guards first as Older Systems Only they often are the critical part Chart Speed Incorrect of the system L Column Problems Incorrect Column Guard Problems Sample Solvent Too Strong System not at Equilibrium Corrective Action Dissolve the sample in mobile phase use a weaker diluent or make a smaller injection Use a column heater or cooler unit to stabilize the temperature around the column Allow the column detector etc sufficient time to stabilize If perfor
87. to normal 6 Loosen detector inlet fitting Does thepressure return to normal 7 Loosen column outlet fitting Does the pressure return to normal 8 Loosen column inlet fitting Does the pressure drop to lt 100psi 7 Bar 9 Loosen fitting at guard or in line filter Does the pressure return to normal 10 Loosen injector outlet fitting Does the pressure return to normal 11 Loosen pump outlet fittings Does the pressure return to normal Yes Stabilize the operating environment temperature Set the correct flow rate Refer to the table in section 5 2 1 or your method Calculate check the viscosity Viscous solvents do produce higher system pressures If possible dilute or change to a less viscous solvent mix Go to next question Replace blocked tubing as per the detector manual Flow cell fluid path blockage Refer to the detector manual for cleaning instructions Blocked outlet tubing Replace Voided or blocked column Blocked guard or filter Replace the disposable unit Injector or connecting tubing blocked Unblock as per injector manual instructions Check that the vials are not coring and that samples are particulate free soluble Outlet connecting tubing blocked Replace tubing as per pump operating manual Verify solvent miscibility 12 Pump problem Contact your maintenance provider No Go to next question Go to next question Go to next question Loosen transducer ou
88. tor Serial Number Pump Serial Number Autosampler Serial Number Column Heater Serial Number Other Date Analyst Maintenance Details Appendix D Example Problem Log Sheet Number Issued Detector Serial Number Pump Serial Number Autosampler Serial Number Column Heater Serial Number Other Date Analyst By Problem amp Troubleshooting Outcome 65 Appendix E Example Column History Log Sheet Number Issued By Date Analyst System Suitability Test Results Resolution Tailing Precision Comments Appendix F Example Instrument Use Log Sheet Number Issued Detector Serial Number Pump Serial Number Autosampler Serial Number Column Heater Serial Number Other Date Analyst Method Details By System Clean Up Details 67 References 1 Troubleshooting LC Systems John W Dolan amp Lloyd R Snyder Humana Press 1989 ISBN 0 89603 151 9 2 Foley amp Dorsey Anal Chem 1983 55 730 Trademarks Columbus Phenomenex Inc The following are trademarks of Thermo Fluofix Neos Corp Electron Corporation Genesis Argonaut Technologies Inc Boab ICAT University of Washington BetaMax Inertsil G L Science Inc BETASILY Kromasil The 3M Company BioBasic LiChrosorb EM Industries DECTABONDE Luna Phenomenex Inc Fluophase MetaSil Varian Inc Hypercarb PEEK Vitrex plc TNBERSIP Phenomenex AQUA Phenomenex Inc IEORITET Rheodyne Rheodyne Inc KEYSTONE SEQUEST University of Wash
89. tput fitting set the flow to zero Does the pressure fall to zero Does adjusting the transducer zero the pressure Replace the transducer as per the pump manual Go to next question Go to next question Go to next question Go to next question Go to next question Go to next question Go to next question The most common causes of high pressure are blocked tubing around the injector and column inlet 3 2 2 Low Pressure No Low Pressure Reading Question 1 Is the pump fuse in working order 2 Is the pump on 3 Is there solvent flow 4 Is there solvent in the reservoir 5 Is the low pressure cut off higher than the operating pressure 6 Does solvent flow out of the purge valve when opened 7 Was the pump primed 8 Is air visible in the solvent lines 9 Are the pump heads functioning correctly 10 Is the flow rate set correctly 11 Is the column temperature constant 12 Are there any leaks 13 Is the correct solvent composition being used 14 Was the purge valve closed after priming 15 Is the auto injector in prime mode 16 Is the flow rate delivered the same as the rate entered QUICK TIP The most common causes of no low pressure are the solvent inlet lines not being immersed in solvent no solvent in the reservoir and leaks Yes Go to next question Go to next question Go to question 10 Go to next question Reset the low pres
90. ts please contact your Thermo representative 4 4 4 Blocked Tubing Blocked tubing results in an increase in system pressure deterioration of chromatographic performance and leaks from worn fittings Blocked tubing can either be replaced or the blockage removed If no replacement tubing is readily available the blockage must be removed This can be done in the following way 1 Isolate the blockage as described in section 3 2 1 of this guide 2 Remove the piece of tubing from the instrument reverse it and attach the reversed end directly to a pump 3 Flush the tubing at approximately 0 5 mL min with a suitable solvent to remove the blockage If the material blocking the tubing is not known use a 50 aqueous methanol solution 4 There are occasions when this flush procedure will not remove the blockage for example if it is due to a large insoluble piece of seal etc In such circumstances increase the flow rate to between 3 and 5 mL min to force the blockage out This flush should be used as a last resort because of the risk of formation of solvent aerosols as the tubing unblocks If this flush is used ensure that the waste end of the tubing is placed in a covered beaker or sealed waste bottle for safety 5 If the tubing remains blocks after flushing then it must be replaced Single and Double end SLIPFREE fittings 37 4 4 5 Old and Leaking Fittings Compression and finger tight fittings have a fin
91. unded where necessary The detector should be isolated from all sources of radio interference or cycling equipment for example large electric motors The detector should be adequately grounded If necessary move the detector away from the source of interference or position it within a Faraday Cage Re set to a lower value on the data handling device ECD Only Refer to the detector maintenance manual for repair or replacement instructions Replace electrode filling solution and frit Polish clean the working electrode If the problem remains replace the working electrode 3 3 3 Cyclic Noise Detector Related Problems and Others AU Time Temperature Fluctuations AU Time Time Cycling Equipment Insufficient Stabilization Possible Cause Sort Term Cycling Equipment or Radio Interference Long Term Detector Temperature Problems Ambient Temperature Fluctuations Baseline Drift Unstable Detector Baseline Drift Ambient Temperature Change Baseline Drift Contaminated Detector Flow Cell Baseline Drift Dirty Reference Electrode Baseline Drift Scratched or Contaminated Reference Electrode Noise Spikes Detector Lamp Malfunction Noise Spikes Cycling Equipment and Radio Interference Noise Spikes Detector Electronics Problems Corrective Action Refer to the previous table The heater cycles on and off to maintain the detector temp
92. wing the guidelines in section 5 6 of this guide If the problem remains after cleaning replace the column Contaminated Guard Replace contaminated guard and check the system performance It should improve If there is no improvement check for column contamination as previously discussed 807 Gradient 1 07 Gradient 2 D e o o 1 1 Response MilliVolts a o Peak removed by allowing 10 E a a E 0 5 10 15 20 Time Minutes Gradient 1 Gradient 2 0 0 20 20 25 25 26 30 8 4o more time for final step P a0 E 104 0 o 715 186 T 7 31 10 rea rl t TTT FA 25 30 0 5 10 15 20 25 30 Time Minutes A B Column 5 ym BetaBasic C18 150 x 4 6 mm 90 10 Solvent A 0 1M Ammonium Acetate pH 4 7 0 100 Solvent B Acetonitrile 0 100 Flow Rate 1 mL min 90 10 Detector UV at 254nm Peaks Misidentified Possible Cause Data Handling Inaccuracies Peak Retention Time Variation Corrective Action If a data handling system identifies your peaks then ensure that all the peak retention time variables such as peak windows threshold integration and retention times are correctly entered into sample and calibration tables etc Make any necessary changes and perform a standard injection to ensure that peaks are now correctly identified Refer to the data handling operators manual for further details Refer to sections 3 4 1 to 3 4 3 3 6 Quantitative Results Quantitative assays
93. y is poor consider changing one or all to give a miscible mix Refer to section 5 3 for a solvent miscibility table If the solvent is mixed manually ensure that it is filtered and thoroughly degassed before use The solvent line and pump should be thoroughly primed with this solvent to remove all traces of previous solvents and air The column should have a minimum of 10 column volumes of solvent passed through it to allow equilibration Finally a series of standard injections should be performed to ensure that the system is performing reproducibly If the retention times are reproducible the problem was due to insufficient preparation of the solvents and system If the solvent is mixed automatically from two or more reservoirs follow the same procedure as listed above but this time more solvents will require filtering more lines priming etc It is also advisable to mix the solvents manually and repeat the procedure to check whether there is a problem in the pump mixing proportioning cell Automatic Mixing If the retention times are reproducible the problem was due to insufficient preparation of the solvents and system If the times remain erratic perform the manual mixing analysis Manual Mixing If the retention times are reproducible there is a problem in the mixing proportioning unit of your pump Refer to the pump mainte nance manual for repair cleaning instructions Note If the system is used for multiple methods ensur
94. yum in diameter BioBasic Phenyl 5 um spherica yes use for peptides proteins and other biomolecules Hypersil BDS Phenyl 3 and 5 um spherica yes use for basic compounds BetaBasic Phenyl 3 and 5 um spherica yes 150 appropriate for most applications BETASIL Phenyl 3 and 5 um spherica yes 100 appropriate for most applications Hypersil Phenyl 3 5 and 10 um spherica no use for acids and neutrals Hypersil Phenyl 2 5 and 10 um spherica yes use for acids neutrals and slightly basic compounds L12 A strong anion exchange packing made by chemically bonding a quaternary amine to a solid silica spherical core 30 50 um in diameter L13 Trimethylsilane chemically bonded to porous silica particles 3 10 pm in diameter BETASIL C1 5 um spherical yes 100A appropriate for most applications Hypersil SAS 3 5 and 10 um spherical no use for acids and neutrals L14 Silica gel 10 pm in diameter having a chemically bonded strongly basic quaternary ammonium anion exchange SAX coating Partisil SAX 10 um irregular no 80 pore size L15 Hexylsilane C6 chemically bonded to totally porous silica particles 3 10 pm in diameter BETASIL C6 3 and 5 um spherical yes 100A appropriate for most applications L16 Dimethylsilane C1 chemically bonded to porous silica particles 5 10 ym in diameter L17 Strong cation exchange resin consisting of sulfonated cross linked styrene divinylbenzene copolymer in the hydrogen form 7 11 ym in diameter Hyper

Successful HPLC Operation

Contents

Download Pdf Manuals

Related Search

Related Contents