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| HPLC Troubleshooting |
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| We can help you. |
Click on the respective observation to display the possible causes
and their prevention / remedies.
General instructions for column maintenance and individual rinsing procedures can be found in the accompanying column leaflet or on the corresponding website. |
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Problem area: Peaks
Observation: Broad peaks
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Possible causes
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Prevention / suggested remedy
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1. injection volume too large.
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1. inject smaller volumes or reduce solvent strength for injection to focus the sample components. |
| 2. poor column efficiency. |
2. use mobile phases of lower viscosity, elevated column temperature, lower flow rate or a packing with smaller particle size. |
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3. extra column volume of the LC system too large.
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3. use zero dead volume fittings and connectors; use smallest possible tubing diameter (<0.25 mm) and matched size of fittings.
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4. volume of detector cell too large.
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4. use smallest possible cell volume for the sensitivity required; use a detector without heat exchanger in the system.
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5. detector time constant too slow.
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5. adjust the time constant to the peak width.
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6. sampling rate of the data system is too low.
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6. increase the sampling rate.
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| 7. only some peaks broad: late elution of analytes from a previous run. |
7. flush the column with a strong eluent after each run, or end gradient at a higher concentration. |
| 8. retention times too long. |
8. use gradient elution or a stronger mobile phase for isocratic elution. |
| 9. viscosity of mobile phase is too high. |
9. increase column temperature or use a solvent of lower viscosity. |
Observation: Fronting
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Possible causes |
Prevention / suggested remedy
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1. column overload.
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1. decrease sample amount; increase column diameter. |
2. formation of channels in the column.
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2. buy a new column or have the column repacked. |
Observation: Tailing
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Possible causes |
Prevention / suggested remedy
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1. basic analytes: interactions with silanol groups.
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1. use silica-based base deactivated RP phases (e.g. NUCLEODUR® Gravity, HTec, Isis, Pyramid); switch to polymer-based columns
(e.g. NUCLEOGEL® RP).
silica-based column: silanol interactions.
decrease the pH value of the mobile phase to suppress ionization of the silanol groups; increase the buffer concentration; derivatize the sample to avoid polar interactions. |
2. sample components which can form chelates: metal traces in the packing.
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2. only use high-purity silica-based packings (e.g. NUCLEODUR®) with their very low metal contamination; add EDTA or another chelating compound to the mobile phase; switch to polymer-based columns (e.g. NUCLEOGEL® RP). |
3. dead volume at the column head /
compressed column packing. |
3. avoid pressure pulses; replace the deteriorated column, or, if possible, open the upper endfitting and fill the void with the column packing or some silanized glass fibre wadding; for preparative HPLC: with our VarioPrep columns you can compensate dead volumes with the adjustable end fitting.
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| 4. system dead volume. |
4. minimize the number of connections; use zero-dead-volume connectors; use new capillaries to column/ to detector; check whether all fittings are tight. |
| 5. silica-based column: degradation at high temperatures. |
5. use temperatures below 50 °C.
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Observation: Double peaks
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Possible causes |
Prevention / suggested remedy
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| 1. column overload. |
1. decrease sample amount; increase column diameter. |
| 2. injection solvent too strong. |
2. use a weaker solvent for the sample or a stronger mobile phase. |
| 3. sample volume too large. |
3. if the sample is dissolved in the mobile phase, the injection volume should be smaller than one-sixth of the column volume. |
| 4. dead volume or formation of channels in the column. |
4. replace the column or, if possible, open the upper end fitting and fill the void with the same packing; have the column repacked. |
5. simultaneous elution of an interfering substance.
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5. use sample clean-up or fractionation prior to injection (e. g. SPE with
CHROMABOND® or CHROMAFIX®); improve selectivity by choice of another mobile or stationary phase. |
| 6. simultaneous late elution of a substance from a previous run. |
6. flush the column with a strong eluent after each run, or end gradient at a higher concentration. |
Observation: Negative peaks
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Possible causes |
Prevention / suggested remedy
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1. RI detector: refractive index of the analyte lower than that of the mobile phase.
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1. reverse detector polarity to obtain positive peaks. |
2. UV detector: absorption of the analyte lower than absorption of the mobile phase.
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2. use a mobile phase with lower UV absorption; if recycling solvent, use fresh HPLC grade eluent when the recycled mobile phase starts to affect detection. |
Observation: Ghost peaks
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Possible causes |
Prevention / suggested remedy
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1. contamination.
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1. only use HPLC grade solvents; flush the column to remove impurities. |
2. late elution of an analyte from a previous run.
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2. flush the column with a strong eluent after each run, or end gradient at a higher concentration. |
| 3. unknown interfering substances in the sample. |
3. use sample clean-up or fractionation prior to injection (e. g. SPE with
CHROMABOND® or CHROMAFIX®). |
| 4. RP chromatography: contaminated water. |
4. check the suitability of the water by passing different amounts through the column and measure the peak height of the impurity as a function of enrichment time; use HPLC grade water or purify the water by running it through an old RP column. |
| 5. peptide mapping: oxidation of trifluoroacetic acid. |
5. prepare fresh trifluoroacetic acid solution daily; add an antioxidant. |
| 6. ion pairing chromatography: disturbed equilibrium. |
6. prepare the sample in the mobile phase; reduce the injection volume. |
Observation: Spikes
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Possible causes |
Prevention / suggested remedy
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| 1. air bubbles in the mobile phase. |
1. degas the mobile phase; install a back pressure restrictor at the detector outlet; ensure that all fittings are tight. |
2. column was stored without endcaps.
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2. always store columns tightly capped; flush RP columns with degassed methanol. |
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Problem area: Baseline
Observation: Baseline drifting to higher absorption
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Possible causes |
Prevention / suggested remedy |
1. with gradient elution: strong UV absorption of the increasing mobile phase component B.
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1. use a higher wavelength of the UV detector; use non-UV-absorbing HPLC grade solvents for your mobile phases;
if a UV-absorbing solvent is inevitable, use a UV-absorbing additive in mobile phase A. |
2. accumulation and elution of impurities.
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2. use sample clean-up or fractionation prior to injection (e. g. SPE with
CHROMABOND® or CHROMAFIX®); use only HPLC grade solvents; clean the contaminated column with a strong solvent. |
| 3. viscosity of mobile phase is too high. |
3. increase column temperature or use a solvent of lower viscosity. |
| 4. retention times too long. |
4. use gradient elution or a stronger mobile phase for isocratic elution.
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| 5. poor column efficiency. |
5. use mobile phases of lower viscosity, elevated column temperature, lower flow rate or a packing with smaller particle size.
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Observation: Baseline drifting to lower absorption
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Possible causes |
Prevention / suggested remedy |
1. with gradient elution: strong UV absorption of the decreasing mobile phase component A.
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1. use non-UV-absorbing HPLC grade solvents for your mobile phases;
if a UV-absorbing solvent is inevitable, use a UV-absorbing additive in mobile phase B. |
Observation: Undulating baseline and noise
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Possible causes |
Prevention / suggested remedy |
1. wave-like / undulating baseline: temperature changes in the room.
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1. monitor or avoid changes in room temperature; isolate the column or use a column oven; cover the RI detector to protect it from air currents. |
| 2. continuous noise: detector lamp problem or dirty detector cell. |
2. replace the UV lamp or clean the detector cell. |
| 3. periodic: pump pulses. |
3. repair or replace the pulse damper; purge any air from the pump; clean or replace the check valves. |
| 4. random: accumulation of impurities. |
4. use sample clean-up or fractionation prior to injection (e. g. SPE with
CHROMABOND® or CHROMAFIX®); use only HPLC grade solvents backflush contaminated column with a strong solvent. |
| 5. insufficient solvent mixing during gradient elution or isocratic proportioning. |
5. mix by hand, or – if possible – use solvents of lower viscosity; monitor proportioning precision by spiking one solvent with a UV absorbing substance and measure the resulting detector output. |
| 6. malfunctioning proportioning valves by gradient elution. |
6. clean or replace the proportioning valve; use partially premixed solvents. |
| 7. spikes: air bubble in detector, mobile phase or pump. |
7. degas the mobile phase; install a back pressure restrictor at the detector outlet; ensure that all fittings are tight; always store columns tightly capped; flush reversed phase columns with degassed methanol. |
| 8. spikes: column temperature higher than boiling point of the solvent. |
8. use lower working temperature. |
| 9. occasional sharp spikes: external electric interferences. |
9. use a voltage stabilizer for your LC system or use an independent electrical circuit for your chromatography equipment. |
10. disturbance at dead time:
air bubbles in the mobile phase. |
10. degas the mobile phase or use premixed eluents. |
| 11. difference in refractive index between injection solvent and mobile phase. |
11. if possible, use the mobile phase as solvent for the sample. |
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Problem area: Retention times
Observation: Decreasing retention times
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Possible causes |
Prevention / suggested remedy |
| 1. column overloaded with sample. |
1. reduce the amount of sample or use a column with larger diameter. |
| 2. loss of bonded stationary phase. |
2. replace column; for silica adsorbents use mobile phases between pH 2 and
pH 8 or switch to phases with higher pH stability (e.g. NUCLEODUR® C18 Gravity) |
| 3. increasing flow rate. |
3. check and – if necessary – adjust the pump flow rate. |
Observation: Increasing retention times
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Possible causes |
Prevention / suggested remedy |
| 1. changing mobile phase composition. |
1. cover the solvent reservoirs to avoid evaporation of volatile solvent component; ensure that the gradient system supplies the proper composition; if possible, mix the mobile phase by hand. |
| 2. decreasing flow rate. |
2. check and – if necessary – adjust the pump flow rate; check for pump cavitation; check for leaking pump seals and other leaks in the system. |
Observation: Fluctuating retention times
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Possible causes |
Prevention / suggested remedy |
| 1. fluctuating column temperature. |
1. ensure that the room temperature is constant; if necessary, thermostat or isolate the column. |
| 2. leaks |
2. see at "leaks" |
| 3. only during first few injections: active groups. |
3. condition the column with concentrated sample. |
| 4. insufficient buffer capacity. |
4. use buffer concentrations above
20 mM. |
| 5. insufficient mixing of the mobile phase. |
5. ensure that the gradient system supplies a mobile phase with constant composition; compare with manually mixed eluents; use partially premixed mobile phases.
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| 6. insufficient equilibration at isocratic separation. |
6. pass 10 to 15 column volumes of mobile phase through the column for equilibration. |
| 7. insufficient equilibration at gradient elution. |
7. increase equilibration time with mobile phase A in order to obtain constant retention times for early peaks, also: pass at least 10 column volumes of eluent A through the column for gradient regeneration.
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| 8. insufficient equilibration at reversed phase ion-pairing chromatography. |
8. increase the equilibration time; in ion-pairing chromatography sometimes 50 column volumes may be required for equilibration; long-chain ion-pairing reagents require more time; if possible, use ion-pairing reagents with shorter alkyl chains. |
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Problem area: Pressure
Observation: No / low or decreasing pressure
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Possible causes |
Prevention / suggested remedy |
| 1. insufficient flow (interrupted, obstructed). |
1. check system for loose fittings. Check pump for leaks, salt buildup, unusual noises. Change pump seals if necessary; loosen the cap on the mobile phase reservoir to avoid underpressure. |
| 2. leak in liquid lines between pump and column. |
2. tighten all fittings; replace defective fittings; tighten the rotor in the injection valve. |
| 3. leaking pump check valve or seals. |
3. clean the check valve; replace defective check valves or seals. |
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Observation: Fluctuating pressure
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Possible causes |
Prevention / suggested remedy |
| 1. air bubbles in the pump. |
1. degas all solvents; flush the solvent with helium. |
| 2. leak in liquid lines between pump and column. |
2. tighten all fittings; replace defective fittings; tighten the rotor in the injection valve. |
Observation: Too high / increasing pressure
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Possible causes |
Prevention / suggested remedy |
| 1. salt precipitation / precipitation of buffer components. |
1. especially in reversed phase chromatography with high proportions of organic solvents in the mobile phase; ensure that the solvent composition is compatible with the buffer concentration; reduce the ionic strength and the ratio organic-aqueous in the mobile phase; premix the mobile phase. |
| 2. contamination at the column inlet |
2.use sample clean-up or fractionation prior to injection (e. g. SPE with
CHROMABOND® or CHROMAFIX®); use an 0.5 µm in-line filter; use guard columns; backflush column with a strong solvent in order to dissolve the impurity; replace plugged inlet frits /guard column. |
| 3. microbial growth in the column. |
3. use a mobile phase with at least 10 % organic solvent; prepare fresh buffer daily; add 0.02 % sodium azide to aqueous mobile phases; for storage equilibrate the column with at least 25 % organic solvent and without buffer. |
| 4. viscosity of mobile phase too high. |
4. use a solvent of lower viscosity or increase the temperature. |
| 5. particle size of packing too small. |
5. use a packing with larger particle size (e. g. 3 μm instead of 1.8 μm).
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| 6. for polymer-based columns: swelling of the adsorbent caused by eluent changes. |
6. use only solvents compatible with the column; check proper eluent composition; consult instructions for use for solvent compatibility; use a column with a higher degree of cross-linking. |
| 7. plugged frit in in-line filter / at column inlet or guard column. |
7. replace the end fitting / the frit or guard column.
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| 8. when the injector is disconnected from the column: plugged injector. |
8. clean the injector or replace the rotor. |
| 9. plugged liquid lines. |
9. systematically disconnect system components from the detector end to the blockage; clean or replace the plugged component.
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Problem area: Miscellaneous
Observation: Lack of selectivity
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Possible causes |
Prevention / suggested remedy |
| 1. detector attenuation set too high. |
1. reduce detector attenuation. |
2. not enough sample injected.
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2. increase amount of sample for injection. |
| 3. sample loss during sample preparation. |
3. use an internal standard for sample preparation and optimize your method. |
| 4. sample loss on column. |
4. use an internal standard and optimize your method. |
| 5. autosampler line blocked. |
5. check the flow and clear any blockages. |
| 6. peaks outside the linear range of the detector. |
6. dilute or enrich the sample until the concentration is in the linear range of the detector. |
7. only during first few injections:
sample absorption in sample loop of injector or column. |
7. condition sample loop and column with concentrated sample. |
Observation: Poor sample recovery
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Possible causes |
Prevention / suggested remedy |
1. adsorption on stationary phase.
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1. increase mobile phase strength to minimize adsorption; for basic compounds use a base deactivated packing like NUCLEODUR® Gravity, HTec, Isis, Pyramid... |
| 2. hydrophobic interactions between stationary phase and biomolecules. |
2. use short-chain reversed phase packings; as an alternative you may use hydrophilic stationary phases or ion exchangers. |
| 3. adsorption of proteins. |
3. use another HPLC mode to reduce nonspecific interactions
(e.g. gel filtration or ion exchange); use a mobile phase containing reagents which enhance solubility of the proteins, strong acids or bases (only with polymer-based columns) or detergents like SDS (Sodium dodecyl sulfate). |
| 4. adsorption on tubing and other hardware components. |
4. use inert tubing and fittings made from e.g. PEEK or titanium. |
Observation: Leaks
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Possible causes |
Prevention / suggested remedy |
| 1. column loses stationary phase. |
1. replace column. |
| 2. serious leaks at column or fittings. |
2. tighten loose fittings or use new fittings. |
| 3. serious leak at detector. |
3. replace defective detector seals or gaskets. |
| 4. serious leak at the injector. |
4. replace worn or scratched valve rotors. |
| 5. serious leak at the pump. |
5. replace defective pump seals; check the piston for scratches and replace piston, if necessary. |
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