System and Method Troubleshooting
Contents
1. 130 Rio Dm ati X ean raje zna CUD T TNO AY IPAS be Pula slu Figura 2 9 Unions Butt Joint ouvoeojloc apuo u 15 Figure 13 Fiy he Zar Daad vluma Ligi 52 ali Dead Volume Occuring in a T I e Unio No Butt Joint Critical Connections Injector Column Column Detector No unions in line filters guard columns Single piece tubing 0 007 and smaller I D FIJUTE Sa Farrule cannot seal properly He lf Dimension X is too long leaks will occur Figure 3b Mixing chamber H Dimension X is too short a dead volume or mixing chamber will occur 14 Eluent Composition Effects on the Column Back Pressure T cP viscosity 0 40 80 C 75 viv in water 12 Guard Columns 2THAES IlpooTaciac Purpose trapping retentive impurities nayi evon GVYKPATODUEVOV akabapolov Disadvantage introduces extra connections in critical zone Sample has 1 impurity How many injections will kill 1 of column surface with 1 sample solution and 10 ul injection volume 1 column surface 2 3 m it could adsorb 0 1 uMole 300 injections will reach this level Guard column always decreases system efficiency Retention time W1 2 Theortical Plates DO Guard No Guard Guard No Guard No Guard 2 743 2 696 1047 0 083 3802 5845 13 Autosampler Column Pump Connections From Pump To Column E Needle Metering Syringe2. MeCN H O MeCN H O MeCN H O MeCN H 0 MeCN b 5 10 15 20 25 min Solvent front uEToOTOo O1aA0TH disturbs phase equilibrium Release of trapped Tayidevuevec impurities 25 Method troubleshooting e Problems are usually related to one of the following aill m xc System Column Sample Mobile Phase 26 System System to system compatibility Differences in configuration detector sequence etc Different dwell volume Detector sensitivity always different Wavelength accuracy Bandwidth Environment effects 27 Sample Avoid particulate in the sample Typical cause of inlet filter clogging Filter Sample filtration can change composition Centrifuge Usually cumbersome duokivnrn Sample vials Type of the vial cap and septa affect contamination and carry over Waters systems require 5 filling of 2 mL vial 28 Troubleshooting seguence Pump Any reciprocal pattern avatrodn eikova on chromatogram Pressure fluctuations Baseline drift possible contamination of the solvent Autosampler Injection marks baseline disturbance Cross contamination Vial fill in Sample level Detector Response baseline noise drift etc Wavelength bandwidth accuracy etc 29 Troubleshooting seguence First check is always the plumbing owAnvwoeic leak flow rate pressure Output chro
3. Wrong connection An A From Pump To Column Sampling Needle Metering Syringe Correct connection Waters system Injection Drawing Sample V3 CLOSES Syringe VFFFFFFFFFFFFFFFFVI FFFFFFFFFFFFFFFFFFI FFFFFFFFFFFFFFFFFFI FFFFFFFFFFFFFFFFFFI FFFFFFFFFFFFFFFFFFI FFFFFFFFFFFFFFFVFFI 444444444444444444 444444444444444444 FFFFFFFFFFFVFFFFFFI FFFFFFFFFFFFFFFFFFI VFFFFFFFFFFFFFFFFVI VFVFFFVVFFFFFFFFFV FFFFFFFFFFFFFFFFFFI FFFFFFFFFFV FrrFFFFFFFFI V1 CLOSED Restrictor Loop Needlewash IN GREEN line Needlewash Pump V4 CLOSED L E 2 o lt N z o vw vw z ES Tt C WO EC FrFVFFVFFFFFFFFFFV FFFFFFFFFFFFFFFFFFI FFFFFFFFFFFFFFFFFFI 444444444444444444 444 444444444444444 444444 444444444 444 VL mi d YELLOW line 15 Waters system Injection Injecting Sample V2 CLOSES V3 OPENS VFFFFFFFVVVVVVVVFFFFI FVVVFFFVVVFVFFFVFVFFVI VFVFFFFYVFVYFFVYFFYTI FFFFFFFFFFFFFFFFFFFFI FFFYFFFFFFFFFFFFFFFFFI VFVFFFFFFFFFFFFFFFFFI VFVFFVVVFFVVFFFVFFFFI VFFFFFVVFVVVVVFVFFFFI VFFFFFVFVFVFFFFVFFFVI VFFYFFFYFFVFFFYYFFYT WFFFFFFFFFFFFFF FFFVFFFFFFVFFFFFFFFFI VVVVFFFVVVVFVVVVVFFVI 44444444444444444444 FFFFFFFFFFFFFFFFFFFV FFFFFFFFFFFFFFFFFFFFI FrrrFFFFFFFFFFFFFFFVI VFFFFFFFFFFFFFFFFFI FrYFFFFFFFFFFFFFFFI VFVFFVVFFFVFFFFFFVI VFVFFFVVVVFVVVFVFVI FFFFFFFVFVFFVFFVVVI VVVYFFFYFFFFFYYFFYTI FFFFFFFFF
4. FFFFFFFFFFFI FFFFFFFFFFFFFFFFFFFVI 44444444444444444444 FFFFFFFVVFFFFFFFFFFFI VFFFFFVVVVFVVVVVVFFFI EEEEEEEEEEEEEE EEE evi FFVFFFVFFFVFFFFVFFYFI FFFFFFFFFFFFFFFFFFFVI FFFFFFFFFFFFFFFFFFFVI VFVFFFVVFFFVFFVYFFYTI VFFFFFFFVFVFFFFFFFFFI VVVVFFVVVVVFFVVVVVFVI VFFFFFFVVVFFFFFFVFFVI FFFFFFFFFFFFFFFFFFFVI FFFFFFFFFFFFFFFFFFFFI VFFFFFFFFFFFFFFFFFFVI VFVFFVVFFFVVFFFFFFYFI VFVFFFFFFVVVVFFVFFFFI VVFVFFVVVVVFFFVVVVFVI VFVVFFVYVFVVFFYYFFTTI FFFFFFFFFFFFFFFFFFFFI FFFFFFFFFFFFFFFFFFFFI FF Fr 44 FF 44 V1 OPENS Loop Restrictor VIL ESSET HILL FEEEEEEE FEEEEEEE WrFFFFFT WFFFFFFY FrrFFFFY prrerrrri WrFFFFFT WFFFFFFF WFFFFFFY FEEFFE WFFFFFFY FrFFFFFY prrererri prrrreevi WFFFFFFY WFFFFFFY WFFFFFFT WFFFFFFT IFrrFFFFY WrFFFFFT WFFFFFFFY WFFFFFFY WrFYFFFY WFFFFFFT FFFFFFFY FrrFFFFY WrFFFFFY Ferrero e 2 a D O z s HHI 1 Sample Diluent Apaiwrnc Effect Sample diluent 1 50 50 MeOH Water 2 80 20 MeOH Water 3 90 10 MeOH Water m o a rt o 4 95 5 MeOH Water utter Citrate pH 4 6 5 100 MeOH f JAN 4A A E xu M Mn db bb d RS 3 Tim a im in Incompatible solvents may cause sample precipitation and column clogging Different eluent pH and composition may cause peak splitting 17 Column Length Column length is a compromise ouuBiBoou c between the efficiency and back
5. System and Method Troubleshooting Yuri Kazakevich Seton Hall University Troubleshooting AvTiueTwrrion MIPOBANHATWV There is no standard troubleshooting procedure General Pattern e Locate the problem by ranking kar ra amp n possible causes e Verify the presence of the most probable cause e f present fix the problem otherwise verify the existence of the next possible cause First try to distinguish System problem or Method problem Method vs System Troubleshooting System Method Parameters Parameters Flow stability Flow rate Backpressure e Eluent EK OUOTIK orriIOdOTTIEON type Clogging aTroppagn Eluent composition Detector problems pH Injection suitability pH modifier Injection volume TPOTTOTTOINTNG type Temperature Injection volume Temperature e Gradient profile System Parameters Simple preliminary verification of system setup can save time Bottle fill in Flush if Backpressure Vial fill in Column type Wavelength Inlet filter solvent Flow stability connections connections Cell volume change gt 15 Check valves cross multiple dale mL contamination detectors connection sequence Sampling rate Critical connections Minimize tubing length System Suitability Available HPLC system set margins Trepi0wpia for column selection 20 ul detector flow cell incompatible with lt 3 mm I D columns 10 ul sample loop incompatib
6. in Chromatographic Conditions Column 15 cm x 0 46 cm Luna C18 2 BEluent 90 Aqueous 10 MeCN Aqueous 15 mM K HPO 7H 0 adj to V pH 1 5 9 with H PO Flow rate 1 ml min Temp 25 C eEnhanced sensitivity is obtained by analyzing aniline in its neutral state 21 Column Equilibration Column equilibrates eciooppozet within 30 min in normal eluent composition range Check retention time stability by injecting standard mixture 3 4 times Very high organic gt 98 or very high aqueous gt 80 need 2 h equilibration at 1 ml min In pure water after 20 h equilibration all analytes elute with void volume Chain collapse No After 20 h of water pumping all organic removed from adsorbent pores Water is not wetting the alkylated hydrophobic surface There is no flow through adsorbent particles only around 22 Solvent Purity How much solvent 0 1 ppm total impurity will contaminate 10 of adsorbent surface Average column 200 m g Assume molecular area of 100 A N moles A N 10042 6 102 Assume average 100 g mole 3 mg total accumulation this comes from 30 L of solvent with 0 1 ppm total purity Column has to be cleaned at least once a week 23 Deeper CDS zm Gradient eHigh pressure vs low pressure mixing System dwell vekpoc volume effect Ima mp 24 J Dolan LC GC V 16 1 16 Column Cleaning md SD 4 dD 4 j 30 4 20 1 HAH D M 0 MeCN H O MeCN H O
7. le with 1 mm I D columns 0 2 ul micro injector is useless for conventional columns Suitability Rule Injection volume lt Cell volume Column Dead Volume 0 65 of the empty column volume System Suitability Injection Volume Column 150 x 4 6 mm C18 V 1 7 ml Efficiency 10 000 t p Eluent MeCN Water 70 30 V Ribenzene 2 2 ml V ana oi O ml Ve Y AV N 16 o m o sea W benzene 700 ul W b a p 584 ul Effect of flow cell volume and sampling rate Response time ota8epa arr kpiornc effect Recorder deflection chart divisions Flow cell volume 20 18 16 14 12 10 8 6 4 2 0 0 Cell volume HPLC System set up WASTE RESERVOIR DETECTOR RECORDER Minimize the volume and connections between autosampler column and detector No guard rrpoorao a no prefilter Tubing amp connections 1560 0025 65um ID Natural 7 000 ps 483 bar 1561 004 100pm ID Bleck 7 000 psi 483 bar 1535 005 12 Sum ID Red 7 000 psi 483 hari 1562 006 150um ID Purple 7 000 ps 483 bar 1536 007 175um ID Yellow 7 000 psl 4 amp 3 ban 1531 010 25mm ID Natural DEMO psi 483 ban 1531B 010 25mm ID Blue 7 000 psl 483 har 1565 015 d mm ID Gray 7 000 psi 483 har 1532 020 50mm ID Crange 7 000 psl 483 ban Figura 3h fel im Chamber Il Dmznz e X z 1 sherk 3 ciTe o rris rk ahama weil Dac dim
8. matogram evaluation e SS o o o o o o o co TESES e N wo c o l flow or detection problem 2 possible injection problem 3 correct chromatogram 30 Troubleshooting seguence to 1 2 3 4 5 6 24 4 19 4 14 4 x p 1 2 3 4 5 6 Analysis of chromatogram Compare with previous results Peak tailing Retention shitt Reverse elution 31
9. pressure Column efficiency is proportional to the column length Specific efficiency of particles per one plate decreases with length increase Length Particle ud Specific m Dia um ur Column Overloading YTrep OPTWON TNANC a 80 1 60 lt lt E g 40 IH a 2 20 0 b 125 1 100 lt 5 75 0 0 0 5 1 0 1 5 2 0 Time min 19 Effect of pH on Aniline pK 9 42 pK 4 58 UV absorbance The mobile phase pH at a constant organic composition may have an effect on an ionizable analyte s UV response At 232 nm there is a decrease in aniline s absorbance as this analyte becomes progressively more ionized A plot of the UV absorbance at a particular wavelength versus the pH of the mobile phase will lead to a sigmoidal dependence The inflection point corresponds to the analyte pK TT ae pH 4 2 pH 3 2 S H 1 3 2 9 Chromatographic Conditions p Column 15 cm x 0 46 cm Luna C18 2 Eluent 90 Aqueous 10 MeCN Aqueous 15 mM K HPO 7H O adj to w pH 1 9 with H PO Flow rate 1 ml min m m x a Temp 25 C Wavelength nm cU Effect of pH on Aniline Retention and UV Nu W un 3 Un w 2 pH 2 be W S J e Q 42 D w pH 5 A wP w pH 6 w See w PH 9 CEE E DE CEEOL ESE ED TCE EE ERE ESE CEC ELE o e CECE TITTET Ae NE COSC a TTT TT eee TTT TTT 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time m
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