Tips and Tricks of HPLC System Troubleshooting Trouble Shooting
Contents
1. Causes Absorbance of sample is less than the mobile phase a Equilibrium disturbance when sample solvent passes through th column a Normal with Refractive Index Detectors it Agilent Technologies 26 Ghost Peaks Ghost Peaks Peaks which appear even when no sample is injected Problem Dirty Mobile Phase 20 100 MeOH Gradient No Sample Injected t Agilent Technologies Noisy Baselines Possible Causes Dirty Flow Cell Detector Lamp Failing Pulses from Pump if Periodic Temperature Effects on Detector Air Bubbles passing through Detector Agilent Technologies Page 54 27 Drifting Baselines Gradient Elution Temperature Unstable Refractive Index Detect Contamination in Mobile Phase Mobile Phase Not in Equilibrium with Column Contamination Bleed in System ee Agilent Technologies Chromatographic Results with Wrong Lamp at 214 nm Wavelength TASTE Sipan Raman E LAMP SAPO TROON TOT DY OEM Lamp R Tip Could also be a symptom of aging lamp it Agilent Technologies Page 56 28 Expanded View of Chromatographic Results Generic Source Lamp at 214 nm Wavelength J S N 150 OEM Lamp S N 400 i ho o ps me i BABY Siges 1a Rem one CAME PRPS OO THOT OD S N 300 p3 Peak 1 S N 15 Peak 2 S N 50 Peak 3 S N 50 Lamp from Generic Source 22 io Poor S N2. Column 1 Initial Column 1 Next Day with 1 H PO s Equilibration 5 9 2 3 5 9 Time min Time min The primary analyte was sensitive to mobile phase aging conditioning of the column e The peak shape was a secondary issue metal chelating compound resolved by de activating the active metal contamination Group Presentation Title t Agilent Technologies Agilent Restricted Page 32 September 10 2008Month 16 Metal Sensitive Compounds Can Chelate Hint Look for Lone Pair of Electrons on O or N Which Can Form 5 or 6 Membered Ring with Metal Salicylaldehyde N M K C J 8 hydroxyquinoline a benzoinoxomine 5 membered ring complex 5 membered ring complex t Agilent Technologies Acid Wash Can Improve Peak Shape Before Acid Wash After Acid Wash 50 100 mLs 1 H3PO Columns ZORBAX SB Phenyl 4 6 x 150 mm Mobile Phase 75 25 mM ammonium phosphate buffer 25 ACN Flow Rate 1 0 mL min Temperature RT Sample Size 5 mL e A 1 HPO solution is used on SB columns 0 5 can be used on endcapped columns Agilent Technologies Page 34 17 Example Change in Retention Selectivity Unintended Mobile Phase Variation Tip The Source of the Problem is Often Not the Obvious Change Column 1 Column 2 Column 2 Fresh mobile phase l AN o m 6 4 0 4 Time min Time min Time min I have experimented with our mobile
3. Peak shape can be poor At pH 5 2 91 of the sample exists as the benzoate ion RP retention decreases At pH 3 2 91 of the sample exists as benzoic acid RP retention increases t Agilent Technologies Page 41 Effect of pH on Peak Shape at or Near the Sample pKa Column ZORBAX SB C8 4 6 x 150 mm 5 mm Mobile Phase 40 5 mM KH PO 60 ACN Flow Rate 1 0 mL min Temperature RT CH CHCOOH pH 4 4 pH 3 0 C y CH CH CH Ibuprofen pK 4 4 4 5 6 7 8 3 4 5 6 Time min Time min Inconsistent and tailing peaks may occur when operating close to an analyte pKa and should be avoided Agilent Technologies Page 42 21 Why Worry About pH pH pKa and Weak Bases RN H RaN H Ka R3NH 9 5 CH Ka 1x10 o OCH C 4 phan HOCH CH N CH At pH 9 the sample exists as protonated and unprotonated diphenhydramine in a ratio of 1 1 Peak shape can be poor At pH 10 91 of the sample exists as unprotonated diphenhydramine At pH 8 91 of the sample exists as protonated diphenhydramine it Agilent Technologies pH vs Selectivity for Acids and Bases Column Nucleosil C18 Column mBondapak C18 Mobile Phase 45 ACN 55 phosphate buffer Mobile Phase 60 25 mM phosphate buffer Sample Bile Acids 40 Methanol 1 Salicylic acid 5 2 Phenobarbital 3 Phenacetin 4 Nicotine 5 Methamphetamine Retention J C 111 1975 149 7 12 OC J C 268 1983 1 5 7 EL
4. Inlet Heavy Metals Bad Column Normal Tailing it Agilent Technologies Page 16 Peak Tailing Identifying Column Secondary Interactions Column Alkyl C8 4 6 x 150 mm 5um Mobile Phase 85 25 mM Na HPO pH 7 0 15 ACN Flow Rate 1 0 mL min Temperature 35 C Sample 1 Phenylpropanolamine 2 Ephedrine 3 Amphetamine 4 Methamphetamine 5 Phenteramine No TEA 10 mM TEA USP TF 5 USP TF 5 1 1 29 Lalas 2 1 91 1 18 3 1 63 T 20 4 2 35 1 26 5 WL eee f I 0 0 25 Time min Time min Tip Mobile phase modifier TEA competes with Sample for surface ion exchange sites at mid range pH values Agilent Technologies Page 17 Peak Tailing Low pH Minimizes Secondary Interactions for Amines Column Alkyl C8 4 6 x 150 mm 5um Mobile Phase 85 25 mM Na HPO 15 ACN Flow Rate 1 0 mL min Temperature 35 C Sample 1 Phenylpropanolamine 2 Ephedrine 3 Amphetamine 4 Methamphetamine 5 Phenteramine pH 3 0 pH 7 0 USP TF 5 USP TF 5 4 1 33 4 2 35 Time min Time min Tip Reducing mobile phase pH reduces interactions with silanols and peak tailing Agilent Technologies Page 18 Peak Tailing High pH Eliminates Secondary Interactions for Amines Column ZORBAX Extend C18 4 6 x 150 mm 5mm Mobile Phase See Below Detection UV 254 nm oH 1 Maleate 2 Scopolamine 3 Pseudoephedrine 4 Doxylamine 5 Chlorphenirami
5. makes it difficult to detect low level impurities it Agilent Technologies Page 57 Effect of Detector Response Time The System is operating well the settings were poorly made Slow Data Rates Can Hinder Impurity Detection and Reduce Sensitivity Response Time Agilent 1100 DAD Agilent 1100 WPS with ADVR Column Poroshell 300SB C18 2 1x 75mm 5mm Mobile Phase A 95 H20 5 ACN with 0 1 TFA B 5 H20 5 ACN with 0 1 TFA 1st peak 1 2 sec Flow Rate 2 mL min At 5 pts sec 6 pts Temperature 70 C Detector UV 215 nm Piston stroke 20 1st peak 1 2 sec At 20 pts sec 24 pts sec Sample 1 Neurotensin3 Lysozyme 2 RNaseA 4 Myoglobin e Tip Adjust the response rate of your detector for best peak detection it Agilent Technologies Page 58 29 Conclusions HPLC column problems are evident as e High pressure prevention better than the cure e Undesirable peak shape e Changes in retention selectivity Often these problems are not associated with the column and may be caused by instrument and chemistry issues pH of mobile Phase Instrument Connections Detector Settings Metal Contamination Start With the Correct Questions Find the Answers The Answers will Lead to Solutions it Agilent Technologies 30
6. phase opening new bottles of all mobile phase components When use all fresh ingredients the problem ceases to exist and have narrowed the problem to either a bad bottle of TEA or phosphoric acid Our problem has been solved Group Presentation Title it Agilent Technologies Agilent Restricted Page 35 September 10 2008Month Tip Dwell Volume Differences Between Instruments Can Cause Changes in Retention and Resolution Vp 0 43 mL Column ZORBAX Rapid Resolution Eclipse XDB C8 4 6 x 75 mm 3 5 ym Mobile Phase Gradient 0 100 B in 52 5 min A 5 95 methanol 25 mM phosphate pH 2 50 B 80 20 methanol 25 mM phosphate pH 2 50 Flow Rate 0 5 mL min Temperature 25 C Injection 5S pb Detection 250 nm Sample Mixture of antibiotics and antidepressants Upper trace simulates actual run data entered into DryLab 3 0 software Lower trace is simulated chromatogram for larger Vp Agilent Technologies Page 36 18 Trick Measure and Correct for Dwell Volume Vp If Vbi gt Vo Compensate for longer Vp by adding an isocratic hold to V such that Hold Vp Vp If Vbi lt Vo Delay injection such that Vp delay V it Agilent Technologies Mobile Phase pH and pH Buffers Why Are These So Important in HPLC pH Effects lonization Silica Surface of Column Sample Components of Interest e Buffers Resist Change
7. 1 Desipramine 2 Nortriptyline 3 Doxepin 4 Imipramine 5 Amitriptyline 6 Trimipramine C x10 Competitive C8 Plates Tailing Eclpse XDB C8 USP TF 5 i c High Load Broadening A _ B LHL 5 Tmel min Time min lh Low Load D pi ne WU 0 5 Time min Time tin Tip Evaluate Both Volume and Mass Loading Group Presentation Title Agilent Technologies Agilent Restricted September 10 2008Month Page 22 we ona 11 Peak Shape Broad Peaks All Peaks Broadened e Loss of Column Efficiency e Column Void Large Injection Volume Some Peaks Broadened e Late Elution from Previous Sample Ghost Peak High Molecular Weight Sample Protein or Polymer it Agilent Technologies Unknown Phantom Peaks Column Extend C18 4 6 x 150 mm 5 um Mobile Phase 40 10 mM TEA pH 11 60 MeOH Flow Rate 1 0 mL min Temperature R T Detection UV 254 Sample 1 Maleate 2 Pseudoephedrine 3 Chlorpheniramine 1 Sample 1 Chlorpheniramine maleate Peak 1 maleate 1 Sample 2 Chlorpheniramine maleate and Pseudoephedrine Peak 1 maleate Peak 2 pseudoephedrine Peak 3 chlorpheniramine from 1 injection Plates 1 5922 2 9879 3 779 A Phantom peak from 4 first injection E 0 ary ers Sa i Peninha ai UN 9 10 0 5 10 15 5 Time min Time min Tip The extremely low plates for moderately retained peaks a
8. Tips and Tricks of HPLC System Troubleshooting Agilent Technologies Inc LC Tips And Tricks Seminar Series it Agilent Technologies Trouble Shooting Steps You Have Recognized There is a Problem How Do You Fix It 1S Did System Suitability or Sample Fail 2 d Review Method for Compliance Is The Procedure Being Followed Properly Are Instrument Settings Correct 3 4 Ask More Questions When Did the System Last Function Properly Has Anything Been Changed 4 h Review ALL parameters The Obvious Is Not Always the Cause Was There More Than One Change it Agilent Technologies HPLC System Components Pump Injector Autosampler Column Detector Data System Integrator Problems Can Be Related to All Components in the System it Agilent Technologies Categories of Column and System Problems A Pressure B Peak shape C Retention it Agilent Technologies Pressure Issues Column Observations Potential Problems High pressure Plugged frit Column contamination Plugged packing Low Pressure Leak Flow Incorrect it Agilent Technologies Determining the Cause and Correcting High Back Pressure Check pressure with without column many pressure problems are due to blockages in the system or guard col Remove Column Pressure Still High Remove Guard Pressure Still High f Column pressure is high Back flush column Clear dirty frit s
9. UENT pH e Retention and selectivity can change dramatically when pH is changed Agilent Technologies Page 44 22 Importance of pH and Buffers A Practical Example Why the Sample Dictates Use eWhat Happens When Buffer Used Effectively What Happens When Buffer Ignored or Used Improperly it Agilent Technologies Importance of pH and Buffers A Practical Example Optimized Isocratic Conditions for Cardiac Drugs Column StableBond SB C18 4 6 x 150 mm 5mm Mobile Phase 45 25 mM NaHzPO pH 3 0 55 MeOH Flow Rate 2 0 mL min Temperature 35 C Detection UV 254 nm Sample Cardiac Drugs 1 Diltiazem 2 Dipyridamole 3 Nifedipine 4 Lidoflazine 5 Flunarizine it Agilent Technologies 23 Don t Have Time to Make Buffers or Adjust pH Column StableBond SB C18 4 6 x 150 mm 5 mm Mobile Phase A 20 H20 B 80 MeOH Flow Rate 1 0 mL min Temperature 35 C UV Detection 254 nm Sample Cardiac Drugs Even at very high MeOH Most Components Strongly Retained with Poor peak Shape Due to IEX at a Time min Buffers are critical to good retention and peak shape in many separations it Agilent Technologies Page 47 What If You Work Outside the Buffer Range Columns StableBond SB C18 4 6 x 150 mm 5 mm Mobile Phase A 30 25 mM NaH2PO pH 4 8 unbuffered B 70 MeOH Flow Rate 1 0 mL min Temperature 35 C UV Detection 254 nm Sample Cardiac Drugs 1 Dilt
10. enerated Cellulose RC Recommended Universal hydrophilic membrane compatible with most solvents aqueous and organic High purity extremely low extractables and binding More Uniform Surface Different than Other Cellulose Filters In line Filters Easy to Use and replace Frits Available in 0 2 0 5 and 2 0 Porosity ES ine Much Less expensive than a Column CF ia i lt unt Easier and Faster to Replace than a Column Frit it Agilent Technologies Page 10 What Are Common Peak Shape Issues 1 Split peaks 2 Peak tailing 3 Broad peaks e Many peak shape issues are also combinations i e broad and tailing or tailing with increased retention Symptoms do not necessarily affect all peaks in the chromatogram Each of these problems can have multiple causes it Agilent Technologies Peak Splitting Caused By Disrupted Sample Path Flow Path Disrupted by Void Sample Allowed to Follow Different Paths Through Column Poorly Packed Bed Settles in Use High pH Dissolves Silica IM 4 7 Normal Double Peaks Split or Double Peaks Tip Similar Effect Can be Caused by Partially Plugged Frit it Agilent Technologies Split Peaks from Column Contamination Column StableBond SB C8 4 6 x 150 mm 5 um Mobile Phase 60 25 mM Na HPO pH 3 0 40 MeOH Flow Rate 1 0 mL min Temperature 35 C Detection UV 254 nm Sample Filtered OTC Cold Medication 1 Pseudoephedrine 2 APAP 3 Unknown 4 Chlorphenira
11. iazem Unsuitable Peak Shape 2 Dipyridamole 3 Nifedipine 4 Lidoflazine 5 Flunarizine Agilent Technologies Page 48 24 Don t Forget Match Column to pH of Mobile Phase for Maximum Column Lifetime low pH and high temperature pH 0 8 90 C Zorbax SB C18 Diisobutyl C18 _o Zorbax Rx C18 Dimethy C18 Competitor A C18 Competitor B C18 i Purge Solvent Competitor C C18 50 methanol water with 1 0 TFA A Solute Toluene Competitor D C18 _ Competitor E C18 Relative Retention ti eC aC ii 0 5 000 10 000 15 000 20 000 25 000 30 000 Column Volumes of Mobile Phase Purge Kirkland J J and J W Henderson Journal of Chromatographic Science 32 1994 473 480 eae it Agilent Technologies age 49 Don t Forget Match Column to pH of Mobile Phase for Maximum Column Lifetime High pH and Room Temperature pH 11 RT Mobile Phase 50 ACN 50 Water 0 2 TEA Initial pH 11 After 30 injections pee Ge Tip Use Columns Designed for chosen pH it Agilent Technologies Page 50 Detection Issues Recognize Where the Problem Originates Is it a consequence of technique Is It expected due to use of certain mobile phase components Can it be corrected by adjusting detector parameters e Answers Will Help Find a Solution Let s Explore Some Problems and Solutions gt it Agilent Technologies Peak Shape Negative Peaks Negative
12. in 2 in i Uptight d H 0 080 lt q 0 090 gt i sd In Swagelok it Agilent Technologies 14 What Happens If the Connections Poorly Made Wrong too long Ferrule cannot seat properly t Wrong too short x Mixing Chamber If Dimension X is too long leaks will occur h X If Dimension X is too short a dead volume or mixing chamber will occur E it Agilent Technologies age 29 Stainless Steel and Polymer Fittings Which type is used and when reliable high pressure sealing e Agilent uses Swagelok type fittings with front and back ferrules which give best sealing performance throughout all our LC systems PEEK lt 400b bar System Pressure fittings are ideal where Connections are changed frequently i e connecting columns Pressure is less critical PolyKetone Easy hand tighten column connection 600 bar Pressure Rating PN 5042 8957 10 pk e Fits to SS Tubing Stainless Steel SS fittings are the best choice for pos it Agilent Technologies Page 30 15 Changes in Retention Can Be Chemical or Physical May be caused by e Column aging e Column contamination e Insufficient equilibration e Poor column mobile phase combination e Change in mobile phase e Change in flow rate e Different Gradient Delay Volumes it Agilent Technologies Page 31 Column Aging Equilibration Causes Retention Selectivity Changes Column 1 After Cleanin
13. mine a hisetiona Injection 1 Injection 1 mjecuon After Column Wash with 100 ACN Time min Time min Tine min Tip Column washing eliminates the peak splitting which resulted from a contaminant on the column How could this be prevented Guard Column SPE clean up of samples Periodic column wash Agilent Technologies Split Peaks from Injection Solvent Effects Column StableBond SB C8 4 6 x 150 mm 5 um Mobile Phase 82 H O 18 ACN Injection Volume 30 pL Sample 1 Caffeine 2 Salicylamide 1 A Injection Solvent B Injection Solvent 100 Acetonitrile Mobile Phase 10 Time min Time min Tip Injecting in a solvent stronger than the mobile phase can cause peak shape problems such as peak splitting or broadening Trick Keep Organic Concentration in Sample Solvent lt Mobile Phase Group Presentation Title Agilent Technologies Agilent Restricted September 10 2008Month Page 14 we onn Peak Tailing Broadening and Loss of Efficiency May be caused by Column secondary interactions Column contamination Column aging Column loading Extra column effects it Agilent Technologies Page 15 Peak Shape Tailing Peaks Causes Symmetry gt 1 2 Some Peaks Tail Secondary Retention Effects Residual Silanol Interactions Small Peak Eluting on Tail of Larger Peak Normal Tailing All Peaks Tail Extra Column Effects Build up of Contamination on Column
14. ne 6 Triprolidine 7 Diphenhydramine pH7 H 11 30 20 mM Na HPO 50 20 mM TEA 70 MeOH 70 MeOH Flow Rate 1 0 mL min Temperature RT Time min Tire min Peak Shape and Retention of this pamp l le of basic compounds improves at high pH where column has igh IEX activity Why Er it Agilent Technologies age 19 Peak Tailing Column Contamination Tip Quick Test to Determine if Column is Dirty or Damaged Trick Reverse Column and Run Sample lIf Improved Possible Cleaning Will Help No improvement Column Damaged and Needs to be Replaced O6 test d at QC test after cleaning est forwar QC test reverse direction 100 IPA 35 C direction Plates TF 3 1 7629 2 08 1 7448 2 12043 1 64 Me 2 12237 3 13727 1 69 4 3 15366 47 13355 1 32 2 5 Hga 4 19067 Plates Plates TF 3 25 25 5 0 Time min Time min Time min Column StableBond SB C8 4 6 x 250 mm 5um Mobile Phase 20 H O 80 MeOH Flow Rate 1 0 mL min Temperature R T Detection UV 254 nm Sample 1 Uracil 2 Phenol 3 4 Chloronitrobenzene 4 Toluene it Agilent Technologies Page 20 10 Peak Shape Fronting Peaks ia 7 Normal Fronting ki Symmetry lt 0 9 Causes Column Overload Agilent Technologies Page 21 Peak Tailing Broadening Sample Load Effects Columns 4 6 x 150 mm 5um Mobile Phase 40 25 mM Na HPO pH 7 0 60 ACN Flow Rate 1 5 mL min Temperature 40 C Sample
15. re an indication of a very late eluting peak from a preceding run Agilent Technologies Page 24 12 Extra Column Dispersion Increasing Extra Column Volume Use short small internal diameter tubing between the injector an the column and between the column and the detector Make certain all tubing connections are made with matched fittings a Use a low volume detector cell a Inject small sample volumes ee Agilent Technologies Peak Broadening Extra Column Volume Column StableBond SB C18 4 6 x 30 mm 3 5 um Mobile Phase 85 HO with 0 1 TFA 15 ACN Flow Rate 1 0 mL min Temperature 35 C Sample 1 Phenylalanine 2 5 benzyl 3 6 dioxo 2 piperazine acetic acid 3 Asp phe 4 Aspartame 10 mL extra column 50 mL extra column volume volume tubing T 1 0 Time min Agilent Technologies Page 26 13 Tip Poorly Made HPLC System Connections Can Cause Peak Broadening The System Has Been Optimized and All Tubing Lengths Are Minimum Smallest Diameter Tubing Used Proper Flow Cell Volume Symptom Still Seems to Have Too Much Extra Column Volume What Is Wrong Have You Made the Connections Properly it Agilent Technologies Column Connectors Used in HPLC Troubleshooting LC Fittings Part Il J W Dolan and P Upchurch LC GC Magazine 6 788 1988 ell sd Waters i L_ 0 090 i 10 130 at in giv Rheodyne Tu eu 0 090 TOA o a oa
16. s in pH and Maintain Retention Improve Peak Shape for lonizable Compounds e Effects Column Life Low pH strips Bonded Phase High pH Dissolves Silica it Agilent Technologies 19 Minimize Change in Retention Selectivity Lot to Lot Evaluate e All causes of column to column change e Method ruggedness buffers ionic strength pH sensitivity sample column interactions All causes of column to column change should be considered first especially when only one column from a lot has been tested Page 39 t Agilent Technologies age 39 Lot to Lot Selectivity Change Related to pH Choice pH 4 5 Lot 1 pH 3 0 Lot 1 28 ig of f 3 l I I aaa HL ta atk H I ii a UU 0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 i 14 1 18 Time min Time min pH 4 5 Lot 2 pH 3 0 Lot 2 1 3 A Base i Ade l l A A Ai 3 024 se pew 6 WB ce tee we Time min Time min pH 4 5 shows selectivity change from lot to lot for basic compounds pH 3 0 shows no selectivity change from lot to lot Indication of poorly controlled ionization Group Presentation Title it Agilent Technologies Agilent Restricted Page 40 September 10 2008Month 20 Why Worry About pH pH pKa and Weak Acids RCOO H RCOO H K TRCooHT coo Ka 6 4 x 105 Ht pKa 4 2 At pH 4 2 the sample exists as benzoic acid and the benzoate ion in a ratio of 1 1
17. urface Wash column Eliminate column contamination and plugged packing high molecular weight adsorbed compounds precipitate from sample or buffer O change frit Clear plugged frit PREVENT THIS it Agilent Technologies Column Cleaning Flush with stronger solvents than your mobile phase Reversed Phase Solvent Choices in Order of Increasing Strength Use at least 25 mL of each solvent for analytical columns Mob 100 100 tonitl Must Reverse This Is Time Consuming Acetonitrile 25 Iso to Often Performed Offline Re Equilibrate Tip When using either Hexane or Methylene Chloride the column must be flushed with lsopropanol before returning to your reversed phase mobile phase i Agilent Technologies Changing a Frit May Not Be a Good Idea May not be possible with new generation columns May damage high performance columns Colum Inlet Frit a Do not allow bed to dry Do not touch the column body heat will extrude packing Do nov overtighten TA 3 J Female End Fitting fiale End Fitting Tip Prevention is a Much Better Idea i Agilent Technologies The Trick Prevention Techniques A Better Choice Use column protection In line filters Guard columns Filter samples Filter buffered mobile phases Sample clean up i e SPE Not As Easy Appropriate column flushing it Agilent Technologies Inexpensive Filters Prevent Column Frit Plugging Reg
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