Columns and Supplies for HPLC

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60 Basic HPLC troubleshooting Symptom Possible Cause Solution(s) Baseline disturbance Positive/negative–Difference in Use mobile phase for sample solvent at void time refractive index of injection solvent Detector leaks Catastrophic–Failed detector seal Replace seals/gaskets Drifting baseline Positive direction–Contaminant buildup/elution Flush column, cleanup sample, use pure solvents Negative direction (gradient)– Absorbance of ”A“ mobile phase solvent Use non-absorbing or HPLC-grade solvent Positive direction (gradient)– Absorbance of mobile phase ”B“ Use higher UV wavelength Positive direction (gradient)– Absorbance of ”B“ mobile phase solvent Use non-absorbing or HPLC-grade solvents Random–Temperature changes Insulate column and tubing Random–Temperature changes Thermostat column and tubing Wavy or undulating–Temperature changes in room Monitor room temperature and control Ghost peaks Peaks from previous injection Flush column at end of run with strong solvent Contamination Flush column to remove contaminants Unknown interferences in samples Sample cleanup or pre-fractionation lon pair–Upset equilibrium Prepare sample in actual mobile phase to minimize disturbance Peptide mapping–Oxidation of TFA Prepare fresh daily; use anti-oxidant Reversed phase–Contaminated water Check suitability of water by running different amounts through reversed phase column and measure peak height with elution; use HPLC grade Spikes–Bubbles in solvent De-gas solvents High column Column blockage with irrev. adsorbed Better sample cleanup; use guard column backpressure sample Too high mobile phase viscosity Use lower viscosity solvents or higher temperature Particle size too small Use larger dp packing Plugged inlet frit Replace end fitting Plugged inlet frit Reverse solvent flow Leak Subtle; white powder at fitting– Loose fitting Tighten fitting, cut tubing, or replace ferrule Leak, injection valve Catastrophic–Worn valve rotor Replace rotor in valve Leak, column or other fittings Catastrophic–Loose fittings Tighten or replace fitting Leak, pump Catastrophic–Pump seal failure Replace pump seal
Symptom Possible Cause Solution(s) Negative peaks RI detector–solute refractive index less than solvent No problem; reverse polarity to make positive UV detector–solute absorbance less Use mobile phase with lower UV absorbance; do not than mobile phase recycle solvent too long Noisy baseline Random–Contaminant buildup Flush column; cleanup sample; use HPLC-grade solvents Continuous–Detector lamp problem Replace UV lamp (lasts 1000 hrs) Occasional–External electrical interference Use voltage stabilizer for LC system Spikes–Bubble in detector De-gas mobile phase or back pressure regulator Peak doubling Sample volume too large Injection volume should be 1/6 when mobile phase used for injection Injection solvent too strong Use weaker injection solvent or mobile phase Blocked frit Replace and use 0.5 µm porosity in-line filter Column void or channeling Fill in void with glass beads or packing; repack column Unswept injector flowpath Replace injector rotor Void at head of column Top off column with packing or glass beads Column overloaded with sample Use higher capacity stationary phase Column overloaded with sample Increase column diameter Column overloaded with sample Decrease sample size Single peak–Interfering components Sample cleanup; prefractionation Peak tailing Beginning of peak doubling See peak doubling Unswept dead volumes Minimize number of connections Unswept dead volumes Ensure injector seal is tight Unswept dead volumes Ensure fittings are properly seated Basic compounds–Silanol interaction Switch to polymeric phase Basic substances–Silanol interactions Use stronger mobile phase or add competiting base (e.g. TMA) Silica-based–Column degradation Use speciality column; polymeric column, or sterically protected Silica-based–Silanol interaction Add salt to mobile phase to increase buffer concentration Silica-based–Silanol interaction Lower mobile phase pH to suppress silanol ionization Silica-based–Silanol interaction Derivatize solute to change polar interactions 61
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Columns and Supplies for HPLC
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HPLC columns and supplies Your link
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Free of charge application primer:
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Agilent Technologies cartridge colu
Page 9 and 10: Zorbax guard cartridge The ZORBAX C
Page 11 and 12: Zorbax preparative guard-column har
Page 13 and 14: ZORBAX StableBond guard cardridges
Page 15 and 16: ZORBAX Bonus-RP reversed phase HPLC
Page 17 and 18: Rx guard cartridges 1 (80 Å) Inner
Page 19 and 20: Standard fitting MicroBore columns
Page 21 and 22: Reversed phase columns Hypersil rev
Page 23 and 24: LiChrosorb Reversed phase HPLC colu
Page 25 and 26: LiChrospher Normal phase HPLC colum
Page 27 and 28: PLgel Individual Pore Sized GPC Col
Page 29 and 30: ZORBAX 300 Extend-C18 columns Inner
Page 31 and 32: Columns for biomolecules - ion exch
Page 33 and 34: Special applications columns ZORBAX
Page 35 and 36: Method Development Kits Item Part N
Page 37 and 38: PEEK tubing Description i.d. 0.02 i
Page 39 and 40: Microvials Volume Cap size Descript
Page 41 and 42: Agilent Technologies’ electronic
Page 43 and 44: Syringe filters (All membrane filte
Page 45 and 46: Parts for 1100 Pump modules Descrip
Page 47 and 48: Parts for Capillary LC applications
Page 49 and 50: Connecting capillaries for the 1100
Page 51 and 52: Parts for autosampler Description P
Page 53 and 54: Parts for detectors Description Par
Page 55 and 56: Pharmaceutical Title Publication no
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Page 59: Food and Flavors Title Publication
Page 63 and 64: Symptom Possible Cause Solution(s)
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Columns and Supplies for HPLC

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