866 TROUBLESHOOTING Table 17.3 Leaks (Section 17.4.1): Symptoms and Sources Location Sub-location/Symptom Possible Source Solution Pre-pump (17.4.1.1) Fitting Loose fitting Tighten or replace Proportioning manifold (low-pressure mixing) Loose or damaged valve Tighten valve or replace manifold Pump (17.4.1.2) Fittings Loose or damaged Tighten or replace Check valve Loose Tighten Cross-threaded Replace; may require new pump head Seal damaged Replace Pump seal Worn seal Replace Auxiliary component Loose fitting; damaged component Tighten or replace High-pressure fitting (17.4.1.3) Stainless steel Loose or contaminated Tighten 1/4-turn; clean or replace PEEK Loose or contaminated Turn off pump, reseat tube, retighten; not for use > 6000 psi Autosampler or manual injector (17.4.1.4) Fittings Loose or damaged See fittings adjustments, Sections 17.4.1.1, 17.4.1.3 Low-pressure needle seal Worn or loose seal Adjust or replace High-pressure needle seal Worn or damaged seal Adjust or replace Cross-port leakage Damaged rotor seal Clean, replace rotor seal; may require stator replacement. External leaks not at fittings Worn rotor seal Clean, replace rotor seal Insufficient rotor-to-seal pressure Adjust Seal-pack failure (Waters only) Worn or damaged seals Service or replace 17.5 TROUBLESHOOTING TABLES 867 Table 17.3 (Continued) Location Sub-location/Symptom Possible Source Solution Column (17.4.1.5) Tube connections Loose or damaged See fittings adjustments, Section 17.4.1.3 End-fitting Loose or damaged Tighten; may require new column End-fitting on cartridge column Loose or damaged fitting; damaged seal Tighten; replace seal; may require new assembly Detector (17.4.1.6) Connecting fittings Loose or damaged See fittings adjustments, Sections 17.4.1.1, 17.4.1.3 Cell leaks Loose fitting Tighten Over-pressure damage Remove source of excessive pressure Failed window seal Adjust or replace Non-UV detector See Chapter 4 and operator’s manuals Table 17.4 Pressure Problems (Section 17.4.2): Symptoms and Sources Symptom Location/Symptom Possible Source Solution Pressure too high (17.4.2.1) General Wrong mobile phase; temperature too low; flow rate too high; wrong column; wrong particle size Use correct conditions Blocked tubing Buffer precipitation; sample particulates Replace tubing; remove problem source Blocked or partially blocked frit In-line filter Replace frit; consider additional sample cleanup Guard column Replace guard column Analytical column Reverse column (if permitted); replace column; consider using in-line filter Upper pressure limit mid-gradient Normal gradient pressure changes Adjust upper pressure limit to accommodate (continued overleaf) 868 TROUBLESHOOTING Table 17.4 (Continued) Symptom Location/Symptom Possible Source Solution Pressure too low (17.4.2.2) General Wrong mobile phase; temperature too high; flow rate too low; wrong column Use correct conditions Lower pressure limit Insufficient mobile phase Refill reservoir Overly sensitive limit switch Disable lower pressure limit Massive leak Isolate and fix (Section 17.3.1) High-pressure fitting Loose or contaminated fitting Tighten, clean, or replace Low-pressure fitting Loose or contaminated fitting Tighten, clean, or replace Low-pressure mixing manifold Air leak from unused solvent line; leaky proportioning valve Prime unused lines with organic solvent; replace faulty proportioning valve Inlet check valve Sticking with ACN mobile phases Sonicate in MeOH; replace Pump starvation (failed siphon test) Blocked inlet-line frit Replace Faulty proportioning valve Replace Pinched or blocked tubing Clear or replace Bubbles in pump Inadequate degassing or purging; degasser failure Degas mobile phase and purge pump; replace degasser Pump seal Worn pump seal Replace Pump piston Broken or scratched piston Replace Pressure too variable (17.4.2.3) Bubbles in pump Inadequate degassing or purging Degas mobile phase and purge pump Inlet check valve Sticking with ACN mobile phases Sonicate in MeOH; replace High-pressure fitting Loose or contaminated fitting Tighten, clean, or replace Low-pressure fitting Loose or contaminated fitting Tighten, clean, or replace 17.5 TROUBLESHOOTING TABLES 869 Table 17.4 (Continued) Symptom Location/Symptom Possible Source Solution Low-pressure mixing manifold Air leak from unused solvent line; leaky proportioning valve Prime unused lines with organic solvent; replace faulty proportioning valve Pump starvation (failed siphon test) Blocked inlet-line frit Replace Faulty proportioning valve Replace Pinched or blocked tubing Clear or replace Pump seal Worn pump seal Replace Pump piston Broken or scratched piston Replace Table 17.5 Interpreting Retention-Factor k and Retention-Time t R Changes (Section 17.4.3) k Changes t R Changes Possible Cause More Information No Yes Flow rate (settings, leaks, bubbles, pump problems) Section 17.4.3.1 No Yes Wrong column size Section 17.4.3.2 Yes Yes Mobile-phase error (%B, pH, additives) Section 17.4.3.3 Yes Yes Stationary-phase problem (aging, dirty samples) Section 17.4.3.4 Yes Yes Temperature problem (poor control) Section 17.4.3.5 Table 17.6 Retention-Time Problems (Section 17.4.3.6): Symptoms and Sources Symptom Location/Symptom Possible Source Solution Abrupt change in t R When column changed Wrong column; poor column-to-column reproducibility (especially type-A columns) Use correct column; use columns from same batch; adjust method for more robust separation; use more reproducible columns (e.g., type-B); Section 2.5.4.6, Table 17.5 When mobile phase changed Improperly formulated mobile phase Make new batch of mobile phase; Table 17.5 (continued overleaf) 870 TROUBLESHOOTING Table 17.6 (Continued) Symptom Location/Symptom Possible Source Solution In gradient elution, with change in instrument Different dwell volume Adjust or compensate dwell volume; Section 9.3.8.2 No apparent change in conditions Equipment, bubble, or temperature problem Check for leaks (Table 17.3, Section 17.4.1); check for pressure problems (Table 17.4, Section 17.4.2); check k changes (Table 17.5, Section 17.4.3); Section 2.5.4.6 Retention drifts During first few injections only Slow column equilibration; column ‘‘loading’’ for sample Normal for some columns and samples; allow longer equilibration; ignore first few injections (Section 3.10.2.2); Sections 2.7.1 (general), 7.4.3.2 (ion-pair), 8.5 (normal-phase), 9.3.7 (gradient elution) Over entire sample batch Unstable mobile phase; mobile-phase evaporation; excessive helium sparging Use stable mobile phase; cover reservoir to reduce evaporation; reduce helium sparging time or change to different degassing technique (Section 3.3) Retention too small Only for large-mass injections for some or all peaks, with right-triangle peak shape Mass overload Reduce mass of sample injected; Table 17.11, Sections 17.4.5.3, 2.6.2 All peaks Mobile phase error; flow rate, column, or temperature change See Table 17.5 Some peaks pH problem or other mobile-phase error; column aging Make appropriate adjustments; Section 7.3 (pH); replace column Only for polar solutes Ionic samples Change mobile-phase pH (Section 7.3); use ion pairing (Section 7.4) 17.5 TROUBLESHOOTING TABLES 871 Table 17.6 (Continued) Symptom Location/Symptom Possible Source Solution Neutral or non-ionic samples Try EPG or AQ column; use normal phase (Chapter 8) or HILIC (Section 8.6) Retention too large All peaks Mobile-phase error; flow rate, column, or temperature change; leak See Tables 17.5, 17.3 Some peaks pH problem or other mobile-phase error; column aging Make appropriate adjustments; Section 7.3 (pH); replace column Table 17.7 Peak-Area Problems (Section 17.4.4): Symptoms and Sources Symptom Location/Symptom Possible Source Solution Peak area to large (17.4.4.1) All peaks larger by same proportion Injection volume too large Adjust Sample preparation errors Use correct procedure Detector settings wrong Adjust Different peaks in sample larger by different amounts Detector settings wrong Adjust Peak(s) present in blank Late elution from prior injection Extend run; add strong-solvent flush; modify sample pretreatment Carryover Add autosampler wash steps; change wash solvent; adjust connections; change sample loop material; rearrange injection order Peak area too small (17.4.4.2) All peaks smaller by same proportion Injection volume too small Adjust Sample preparation errors Use correct procedure (continued overleaf) 872 TROUBLESHOOTING Table 17.7 (Continued) Symptom Location/Symptom Possible Source Solution Detector settings wrong Adjust Different peaks in sample smaller by different amounts Detector settings wrong Adjust Peak area too variable (17.4.4.3) Replicate injections give constant areas Problem prior to injection Check sampling and sample pretreatment steps Replicate injections give inconsistent areas Problem from injector onward Check autosampler, pump, detector, and integration Table 17.8 Baseline-Drift Problems (Section 17.4.5.1): Symptoms and Sources Symptom Location/Symptom Possible Source Solution Periodic drift 1 cycle/run Normal with gradient elution Ignore; use higher wavelength; change solvent; add UV absorber Several hours or 1 cycle/day Laboratory temperature cycle Thermostat oven; move system from drafts; adjust lab HVAC Other drift Baseline drifts, then stable Normal equilibration; detector warm-up Wait until baseline stabilizes Table 17.9 Baseline-Noise Problems (Section 17.4.5.2): Symptoms and Sources Symptom Location/Symptom Possible Source Solution Short-term noise High-frequency (50 or 60 Hz) ‘‘Dirty’’ electrical supply; too small of detection time constant; too high of data rate Use UPS to cleanup supply; use larger time constant or RC filter; use slower data collection rate 50–60 Hz < noise < peak width Inadequate mobile-phase degassing Use better degassing technique 17.5 TROUBLESHOOTING TABLES 873 Table 17.9 (Continued) Symptom Location/Symptom Possible Source Solution Long-term noise Cyclic Bubble in pump; piston seal or piston damage; sticking or leaking check valve Degas mobile phase; service pump; clean or replace check valve Appears as chromatographic peak Late-eluted solute Extend run; add strong-solvent flush to method; change sample cleanup Random, irregular baseline disturbances Accumulated late-eluted non-polar materials from sample Add strong-solvent flush to method; replace column; change sample cleanup Peaks appear in blank gradients (‘‘ghost’’ peaks, Figs. 17.11, 17.12) Contaminated mobile phase Replace mobile-phase reagents with fresh and/or higher purity reagents Table 17.10 Peak-Shape Problems (Section 17.4.5.3): Symptoms and Sources Symptom Symptom Symptom/Possible Source Solution Peak tailing TF ≤ 1.2 Normal None required 1.2 < TF ≤ 2 If increased over time: column aging or contamination; mobile phase error Flush or replace column; prepare new batch of mobile phase If constant, may be normal No action or explore solutions for TF > 2 TF > 2 For all peaks Also see split or distorted peaks Early peaks tail more than later peaks (Fig. 17.13): extra-column effects Reduce extra-column volume (Sections 2.4.1, 3.9) (continued overleaf) 874 TROUBLESHOOTING Table 17.10 (Continued) Symptom Symptom Symptom/Possible Source Solution Worse for bases and ionic compounds (Fig. 5.9a): silanol effects; trace metal contamination Adjust pH; use higher purity or less-active column; see Sections 5.4.4.1, 7.3.4.2 Inadequate buffering or mobile-phase additive concentration Increase buffer or additive concentration; see Section 7.2.1.1 Right-triangle peak tailing accompanied by earlier retention (Fig. 17.15a) Mass overload of the column Reduce injection volume or dilute sample for smaller mass on column (Section 2.6.2) Peak fronting TF ≥ 0.9 Normal None required TF < 0.9 For ion pairing, especially with type-A columns Change temperature ±5−10 ◦ Cmayhelp; change to type-B column Column void or bed collapse Replace column; operate column below high pH-limit Broad peaks N > 75% of manufacturer’s test May be normal No action required; compare to new column test with sample solutes High-molecular- weight compounds (proteins, polymers, etc.) Some broadening normal No action required Gradual broadening over > 500 injections Normal column aging Flush column with strong solvent; replace column In the presence of narrower peaks (Fig. 17.5) Late elution Extend run; adjust run length; use gradient flush; improve sample cleanup Accompanied by longer retention times Column temperature too low Increase temperature 17.5 TROUBLESHOOTING TABLES 875 Table 17.10 (Continued) Symptom Symptom Symptom/Possible Source Solution Narrower peaks broadened more than normally wider peaks Detector time constant too large; integrator data-rate too low; excessive smoothing (especially MS detection) Use time constant ≤1/10 peak width; collect a minimum of 20 points across a peak; use less smoothing early peaks broad or flat-topped (Fig. 17.16b) Too large an injection volume Reduce injection volume; dilute injection solvent (Sections 2.6.1, 3.6.3) Split or distorted peaks All peaks distorted in same manner (Figs. 17.17, 17.19) Partially blocked column inlet-frit; column void Reverse-flush column; use in-line filter or better cleanup; replace column (Section 5.8) Broad or distorted peaks, especially at end of run (Fig. 17.20b), accompanied by change in retention time Mismatch of mobile phase and column temperature Use better temperature control; use mobile phase pre-heater (Section 3.7.1) Early peaks distorted (Fig. 17.16c), usually accompanied by earlier retention Too large a volume of too strong a sample-injection solvent Use more dilute injection solvent; reduce injection volume (Sections 2.6.1, 3.6.2.2) Table 17.11 Failed System Performance Tests (Section 17.4.6): Symptoms and Sources Failed Test Symptom Symptom/Possible Source Solution Gradient linearity Steps in gradient Blocked reservoir frit; bad proportioning valve Check/replace frit; replace proportioning manifold Segmented appearance in linear gradient Software control error Adjust control parameters (see service manual) (continued overleaf) . leakage Damaged rotor seal Clean, replace rotor seal; may require stator replacement. External leaks not at fittings Worn rotor seal Clean, replace rotor seal Insufficient rotor -to- seal pressure Adjust Seal-pack. replace Non-UV detector See Chapter 4 and operator’s manuals Table 17.4 Pressure Problems (Section 17.4.2): Symptoms and Sources Symptom Location/Symptom Possible Source Solution Pressure too high (17.4.2.1) General. inconsistent areas Problem from injector onward Check autosampler, pump, detector, and integration Table 17.8 Baseline-Drift Problems (Section 17.4.5.1): Symptoms and Sources Symptom Location/Symptom Possible Source