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Troubleshooting overview
Running into issues with your HPLC or UHPLC analysis? The sections below provide LC troubleshooting help with a list of symptoms, root causes and solutions for common UHPLC and HPLC problems as well as more comprehensive solutions with special technologies such as nano LC, fluorescence and charged aerosol detection. Additional resources include:
HPLC Instrument Manuals ›
The troubleshooting section of each instrument manual provides a specific list of error codes and messages to identify and troubleshoot in detail problems that may arise during the operation of your Thermo Scientific HPLC instruments and associated equipment.
HPLC Knowledge Base ›
Find the latest, complete collection of troubleshooting hints, tips, and tools in our Knowledge Base. Use the Knowledge Base to search for troubleshooting information about retired Thermo Scientific products as well.
Release Notes Chromeleon CDS ›
With Thermo Scientific Chromeleon 7.3.2 Chromatography Data System (CDS), we introduced an in-built troubleshooting tool for Thermo Scientific Vanquish HPLC and UHPLC instruments. It provides solutions to fix error messages and error codes on Vanquish devices connected to your Chromeleon 7.3.1 CDS. If a diagnostic test is available that helps identify the cause or solve the problem, the last test result or status is displayed, and the test can be started directly from the troubleshooting tool in Chromeleon 7.3.2.
Peaks
| Possible cause | Solution |
|---|---|
| Instrument failure, wrong elution conditions | Does the detector show typical baseline noise? If the detector output is just a flat line, the detector or the data transfer failed. Inject known test substance without column and check detector response. |
| No injection, not enough sample volume | Ensure sample is drawn into sample loop. Check that injection has taken place (pressure drop at the beginning of run). |
| High background current/noise (Charged Aerosol Detection, CAD) | Check mobile phase quality. See baseline section. Read the technical note. |
| Sample too volatile (Charged Aerosol Detection, CAD) | Check response by flow injection analysis. Check vapor pressure of analyte(s). Read the technical note. |
| Possible cause | Solution |
|---|---|
| Basic compounds interact with silanol groups | Use type B (high-purity) silica or shield phases (such as polar-embedded groups). Use competing base such as triethylamine (TEA). Alternatively (with type A silica columns), use polymeric columns. Use buffers of high ionic strength (displacement effects – NOT compatible with LC/MS). |
| Insufficient buffer capacity | Increase buffer concentration. |
| Chelation with trace metals in stationary phase | Add competing chelating agent (such as EDTA) to the mobile phase. |
| Extra column volume too large | Use short capillary connections. Check inner diameter of connecting capillaries: 0.13 mm (0.005 in.) for UHPLC columns and 0.18 mm (0.007 in.) for conventional HPLC columns. The extra-column volume should not exceed 1/10 of the smallest peak volume. Use Thermo Scientific Viper or nanoViper fingertight fitting system capillaries. |
| Column degradation | Replace column. With high temperatures: Avoid high temp in combination with aggressive buffers (e.g., phosphate) - reduce to uncritical temperature (e.g., 40 °C), replace column type (e.g., hybrid column). With high pH: Use high pH compatible column type. |
| Column void (particularly at UHPLC pressures) | Replace column. Try to flush reversed column (outlet to waste). Preventative actions: Slowly increase the column flow to avoid pressure shocks on the column. Avoid aggressive pH (check column specifications). Routinely operate columns at less than 70-80% of the pressure specification. |
| Improper capillary connections | Check fittings for correct placement of ferrule. Use Viper or nanoViper Fingertight Fitting System capillaries. |
| Possible cause | Solution |
|---|---|
| Blocked frit, particles on column head | Replace pre-column frit. If fronting returns quickly, try to locate source of particles (sample, eluents, pump mechanics, injection valve). |
| Channels in column | Replace column. Check if application conditions are within the column specifications (e.g., pressure and pH range). |
| Column overload | Reduce amount of sample. Increase column volume (use larger i.d.). |
| Sample dissolved in strong eluent | Dissolve sample in starting mobile phase. Reduce sample solvent strength or injection volume. Read the application notes: Overcoming strong solvent effects during method transfer and Improving peak results using a custom injection program. |
| Possible cause | Solution |
|---|---|
| Detector cell volume too large | Flow cell volume should not exceed 1/10 of the smallest peak volume. Use smaller volume flow cell (i.e., micro or semi-micro) with UHPLC or microbore columns. |
| Early peaks broader than later eluting ones | Extra-column volume is too large. Check capillary i.d. and length, sample loop size, flow cell, etc. |
| Detector response time (time constant) too long | Select a response time less than 1/4 of the peak width at half-height of the narrowest peak. Use Chromeleon Chromatography Data System program wizard to optimize the response time (time constant) settings. |
| High longitudinal dispersion | Isocratic separation: Retention time too long. Use gradient elution or stronger isocratic mobile phase; use less retaining stationary phase (e.g., C8 instead of C18). Linear velocity too slow: Check flow rate. |
| Charged Aerosol Detectors (CAD) compared to e.g., UV detection | Charged aerosol detection will broaden peaks more than UV due to nebulization. |
| Possible causes | Solution |
|---|---|
| Only some peaks broad: Late eluting peak from previous injection | Extend run time. Increase elution strength of gradient (higher organic content). At the end of the run, flush column with strong eluent. |
| Contamination (typically injector or column) | Flush sampler, replace parts prone to contamination (e.g., needle or needle seal). Flush column with strong eluent (also check column instructions; flush in reverse flow direction if possible). |
| Interferences in sample | Use sample cleanup techniques such as solid-phase extraction (SPE). |
| Contaminated nebulizer (Charged Aerosol Detection, CAD) | Nebulizer chamber may need to be cleaned. Remove column. Wash your charged aerosol detector with 50/50 water:methanol at 2.0 mL/min for several hours. Adjust flow-rate to 0.2–0.4 mL/min overnight. Re-check noise level of original mobile phase composition. |
| Contamination of eluents | Water quality: Run different amounts of water over the column and measure enrichment time as a function of the volume. Replace water with HPLC-grade water. Contaminations may also result from bacterial growth in the degasser, eluent modifiers (e.g., degraded TFA) or improper liquid handling (e.g., amino acid mobile phase prepared without gloves). |
| Possible causes | Solution |
|---|---|
| Contamination on column or guard inlet | 1. Replace guard. 2. Flush column with strong mobile phase, backflush to waste (if possible). 3. Replace analytical column. |
| Sample solvent too strong | Prepare/dilute sample in mobile phase. Read the application notes: Overcoming strong solvent effects during method transfer and Improving peak results using a custom injection program. |
| Coelution with unknown interference | Perform efficient sample cleanup. Adjust selectivity by changing the mobile phase or the column. |
| Worn out rotor seal | Replace rotor seal. With extreme pH applications, check compatibility of the seal polymer. |
| Temperature mismatch | Mainly occurs with column inner diameter’s > 3 mm in combination with high column temperatures. Use an eluent pre-heater to ensure eluent temperature does not result in temperature gradient over column. |
| Improper capillary connections | Mainly occurs with column inner diameter’s > 3 mm in combination with high column temperatures. Use an eluent pre-heater to ensure eluent temperature does not result in temperature gradient over column. |
| Packing integrity loss (UHPLC applications) | Replace column. Check pressure rating of the column and work at maximum 70–80% of the pressure spec. Slowly increase pressure on columns to avoid pressure shocks. |
| Possible causes | Solution |
|---|---|
| Absorption/fluorescence of analyte is lower than mobile phase | 1. Change UV/fluorescence detection wavelength(s). 2. Use mobile phase with less background absorption/fluorescence. 3. Dissolve sample in mobile phase. |
| Wrong polarization of analog output interface | Check cable polarity at analog output. |
| Inappropriate reference wavelength setting (DAD) | The sample must not absorb in the range of the reference wavelength. If possible, use a method without reference wavelength. |
| Drainage spiking (Charged Aerosol Detection, CAD) | Ensure that PEEK tubing is present within the drain/vent assembly. |
| The fluorescence of the substance is quenched by matrix or mobile phase (FLD) | 1. Evaluate changes to the composition of the mobile phase. 2. Consider using negative peaks for quantification. |
| Possible causes | Solution |
|---|---|
| Non-ideal fluorescence detector settings (FLD) | Scan for best excitation and emission wavelengths, optimize the gain of the photomultiplier, use high-quality mobile phase only, set suitable response time. |
| Non-ideal UV detector settings (DAD) | Scan for best absorption wavelength(s), set suitable response time and optimize slit widths and bandwidths according to the operating manual. Use appropriate flow cell i.e. Lightpipe 10 mm vs 60 mm. |
| Possible causes | Solution |
|---|---|
| Quenching caused by insufficient degassing | Check degasser operation. |
Peak area precision
| Possible causes | Solution |
|---|---|
| Sample or sampler problem | Perform multiple injections to differentiate between sampler and sample related issues: 1. Variations of the sum of the peak areas → Injector. 2. Only some peak areas vary → Sample not stable. To verify: Inject known stable mixture → peak areas should not vary. 3. If peak areas of some peaks still vary, check 3. system pressure stability and short-term flow stability. |
| Possible causes | Solution |
|---|---|
| Positions of integration delimiters vary | Check the software integration settings. For instance, with the support of the Chromeleon Chromatography Data System help. Best, facilitate the Chromeleon 7 Cobra algorithm and let it identify the ideal settings for you. Avoid automatic data rate settings and use a fixed data rate. |
| Possible causes | Solution |
|---|---|
| Irreproducible integration caused by pump pulsation, mixing ripple, etc. | Please refer to the baseline table, symptom: Periodic Baseline Fluctuation. |
| Possible causes | Solution |
|---|---|
| Autosampler draws air from the vial | Check the sample filling height and the sampling height of the injector needle. |
| Sample degradation | Use appropriate storage conditions, e.g., thermostatted autosampler. |
| Air in the autosampler fluidics | Flush out autosampler fluidics following the steps laid out in the respective operating instructions. |
| Injector needle clogged or needle tip deformed | Replace needle. Remove air from autosampler fluidics. |
| Autosampler draw speed too high, sample with high gas content | Reduce the draw speed to take at least 2-3 seconds. Program a delay time after the drawing of sample (e.g., 3 seconds). Degas sample and/or decrease draw speed |
| Leaking injector seal, bubble in syringe | Check injector seals, purge syringe. |
| The autosampler, the injection valve, and/or the syringe valve are not tight | Check seals and tighten fittings involved in the injection process of the sampler. |
| Possible causes | Solution |
|---|---|
| Wrong detection wavelength(s) | Measuring in a UV/fluorescence spectrum flank can compromise the precision. Choose a detection wavelength or an excitation/emission wavelength pair near the apex of the spectrum/spectra. If spectra of analytes are very different, a wavelength switch might be required. |
| Response time too short, high noise, imprecise integration at trace level | Make sure to use suitable response time (or time constant) settings. Typically set a response time that is about 1/4 of the peak width at half-height of the narrowest peak. Follow the operating instructions for details. |
| Incorrect nebulizer temperature (Charged Aerosol Detection, CAD) | Check nebulizer temperature setting. If using large amounts of THF or halogenated solvents in mobile phase, set temperature to 30 °C. If analyte is semivolatile, it may be necessary to turn off the nebulizer heater to recover response. |
| Not enough data points | Set the data collection rate at least to 20-30 data points for each peak for reproducible peak integration. |
| Possible causes | Solution |
|---|---|
| Carry over (from needle, sample loop, needle seat) | Extend autosampler wash options to reduce carry over. Clean needle, needle seat, and rotor seal. |
| Column memory effect | Run blank sample (no injection) after sample. If a peak is detected, then a column memory effect is present. Flush column with strong eluting eluent. Reverse column (if allowed) and flush column again. Replace column. |
Pressure
| Possible causes | Solution |
|---|---|
| Buffer precipitation | 1. Check buffer compatibility with max organic content (in beaker). 2. Flush out system and column with low organic content. 3. Systematically investigate fluidics to locate the blocked part (from detector to pump). |
| Bent capillaries | Systematically investigate fluidics to locate the blocked part (from detector to pump). |
| Particles on mixer, frit, guard, or column | Use pre-filtered mobile phases suitable for the column particle size (for instance 0.1-0.2 μm for sub-2 μm particles, 0.45 μm for 3–5 μm particles). Filter samples accordingly. Check sealing mechanic parts (piston seal, rotor seal). |
| Column aging/blocking | Gradual pressure increase is normal over the lifetime of a column. Unusual pressure increase: Check connection tubings, temperature control, replace column. |
| Pressure changes with the course of a gradient | Normal. The system pressure is dependent on the viscosity of the mobile phase composition in the system fluidics. |
| Blocked/clogged liquid inlet or nebulizer (Charged Aerosol Detection) | Check backpressure of the charged aerosol detector. Backpressure should be under 10 bar (145 psi). |
| Too high flow or too low column temperature setting | Correct setting. |
| Possible causes | Solution |
|---|---|
| Leaking capillary connections | 1. Check fittings for tightness, particularly before the column. PEEK capillaries with fingertight fittings may slip out of the ideal position. 2. Use Viper or nanoViper fingertight fitting system capillaries for virtually zero-dead-volume connections. |
| Flow too low or column temperature setting too high | Correct setting. |
| Possible causes | Solution |
|---|---|
| Air trapped in pump | 1. Degas eluents, best with on-line vacuum degassing or helium sparging. 2. Purge the pump against a gentle backpressure. 3. Actively draw liquid through the pump, for instance, with a syringe at the outlet of the pump. 4. Temporarily replace the water with degassed organic solvent and purge the respective eluent line. Afterwards, switch back to degassed water. |
| Inlet or outlet check valves of the pumps are dirty | Take out check valve cartridges, place them in a container with a suitable liquid (e.g., methanol) and clean them in an ultrasonic bath. |
| Failing pump check valves or piston seals | High-pressure proportioning: Identify the failing pump block by relating the pressure drops to the pump cycle. Change the % B and observe how the frequency of pressure drops changes. Use Thermo Scientific Chromeleon Chromatography Data System software's pump diagnostics to identify the failing part. |
| Blocked solvent line frit | Replace solvent line frit. |
Retention times
| Possible causes | Solution |
|---|---|
| Degradation of stationary phase | Replace column. Check pH requirements of the column, typically pH 2–8 for silica-based RP columns. |
| Insufficient equilibration | Increase equilibration times, at least 5–10 times the column volume. |
| Column overload | Reduce amount of sample. Increase column volume (use larger i.d.). |
| Increase flow rate | Check flow rate setting, perform OQ on flow rate precision. |
| Stationary phase dewetting at low organic content | Use stationary phase compatible with low organic content (e.g., polar embedded phases). Re-wetting the column is facilitated by high organic content at increased pressure. Check column care manual for details. |
| Mobile phase composition | 1. Check premixed mobile phase, make sure the liquid is homogeneous. 2. Perform an OQ proportioning test. 3. a) High-pressure proportioning: Check for leaks or bubbles in aqueous eluent. b) Low-pressure proportioning: Check/adjust proportioning valve. |
| Possible causes | Solution |
|---|---|
| Mobile phase composition | 1. Check premixed mobile phase, make sure the liquid is homogeneous. 2. Perform an OQ proportioning test. 3. a) High-pressure proportioning: Check for leaks or bubbles in aqueous eluent. b) Low-pressure proportioning: Check/adjust proportioning valve. |
| Active sites on stationary phase | Use organic modifier base (such as TEA), increase buffer strength or use higher coverage column packing. |
| Decreasing flow rate | Leaking capillary connections. Check flow rate settings. |
| Pre-column dead volume (mainly nano/cap applications) | If peaks are late, but not broad there is a dead volume before the separation column. If the peaks are broad and late the dead volume is after the column. |
| Possible causes | Solution |
|---|---|
| Insufficient equilibration | Increase equilibration times, at least 5–10 times the column volume. |
| Imprecise eluent proportioning | Perform an OQ eluent proportioning test. Clean/replace pump check valves, or call service for adjusting/replacing proportioning valve. |
| Insufficient buffer capacity | Increase buffer concentration > 20 mM. Work at pH within buffering range. |
| No synchronization of injection with pump cycle (low-pressure proportioning) | Activate function in the software, for instance in the Chromeleon Chromatography Data System’s pump driver. |
| Changing column temperature | Place column in column thermostat. Monitor oven temperature. |
Baseline
| Possible causes | Solution |
|---|---|
| Pressure fluctuations from pump | Record a pump pressure channel to identify temporary or permanent pressure ripple. A pump influence is indicated by baseline fluctuations synchronous with the pump cycle. Purge the pump, check general functionality (for instance with a diagnostics test), read pre-compression value from pump panel and compare against values from manual (Thermo Scientific pumps only). Check that appropriate mixing volume for applied eluents/flow rate is selected. |
| Air in the pump fluidics | 1. Degas eluents, best with on-line vacuum degassing or helium sparging. 2. Purge the pump. 3. Actively draw liquid through the pump, for instance with a syringe at the outlet of the pump. 4. Temporarily replace the water with degassed organic solvent and purge the respective eluent line. Afterwards, switch back to degassed water. |
| Wrong reference wavelength selected (DAD) | The sample must not absorb in the range of the reference wavelength. If possible, use a method without reference wavelength. |
| Insufficient mixing | Periodic, wave-like noise. Intensity of effect is related to the eluents, the type of detector, and the detector layout. Increase the mixing volume depending on the manufacturer‘s recommendation. |
| Improper grounding | The complete HPLC system should be plugged into the same circuit. Isolate the electrical circuit from strong current consumer devices or use an isolation transformer to filter current fluctuations. |
| Absorbing solvent | Check mobile phase for degradation, particularly aqueous buffers. With DADs, switch off the reference wavelength during troubleshooting. Prepare fresh mobile phase. |
| Improper installation of optical components | Check if lamps and flow cell are positioned properly. |
| Mobile phase degraded | Prepare fresh mobile phase, and flush the system with it. If required, backflush the column (increased backpressure). |
| Possible causes | Solution |
|---|---|
| Mobile-phase quality | Make sure to use HPLC-grade or better mobile phase quality, particularly for sub-2 μm particle columns and evaporative detectors (such as the Charged Aerosol Detector, CAD) use mobile phase with lowest particle content whenever possible. |
| Response time (time constant) setting too short → increased noise | Make sure to use suitable response time. Typically set a response time that is about 1/4 of the peak width at half-height of the narrowest peak. Follow the operating instructions for details. |
| Optical bandwidth setting (UV detectors) too narrow | Use a low optical bandwidth setting for best optical resolution. Increase the bandwidth setting for best signal to noise. Please refer to the detector‘s operating instructions for details. |
| Nonvolatile mobile phase (Charged Aerosol Detection) | Make sure to use volatile buffers and additives with Charged Aerosol Detection. Read the technical note. |
| Nebulizer (Charged Aerosol Detection) | Nebulizer chamber may need to be cleaned. Remove column. Flush your charged aerosol detector with 80/20 water:methanol at 2.0 mL/min for several hours. Adjust flow-rate to 0.2–0.4 mL/min overnight. Re-check noise level of original mobile phase composition. |
| Nitrogen gas supply (for evaporative detectors) | Make sure to use 99% pure nitrogen, free from water vapor and particulates. Replace two-stage filter if necessary. |
| Lamp is too old (optical detectors) | Check lamp age and check number of ignitions. Replace the lamp if needed. If after renewing the lamp the noise is high check that the lamp has been installed properly. |
| Possible causes | Solution |
|---|---|
| Detector lamp/optics temperature not stable | Allow the detector to warm up. Depending on the optical design, this may take 30 minutes to a few hours. See the detector‘s operating instructions for details. |
| Mobile phase not homogeneous | After a day or more idle time, gently swirl the eluent bottle to homogenize eluents already in their reservoir. |
| Column contamination | Flush the column with strong eluent. If possible, try to reverse the column (check with column specifications). |
| Causes as discussed in non-periodic, high noise (Charged Aerosol Detection) | Solutions as discussed in non-periodic, high noise Charged Aerosol Detection |
| Gradient operation (Charged Aerosol Detection) | Minimize gradient baseline effects by using appropriate mobile phase quality. Use shallow gradient methods whenever possible. Use inverse gradient. Read the technical note. |
| New detector lamp | For a few hours, allow the lamp to reach a constant light intensity |
| Non-constant ambient conditions | Make sure that the temperature and the humidity in the laboratory are constant. With Chromeleon Chromatography Data Systems software, record temperature fluctuations with the help of detector temperature channels. Verify that the lamp and flow cell covers are in their proper position and that the front panel door is closed. |
| Contaminated flow cell | Clean the flow cell (refer to the operating instructions for a recommended procedure). If necessary, replace the flow cell. |
| Possible causes | Solution |
|---|---|
| Ion-pairing application | Normal. Ion pairing applications always require long initial equilibration times. Shorter alkyl-chain lengths of the ion-pairing agent allow for less equilibration than longer chains. |
| Possible causes | Solution |
|---|---|
| Air inside flow cell fluidics | Typically, bubbles appear randomly. Identify by: 1. Observing similar spike heights at different wavelengths Or 2. Applying a slight backpressure on the flow cell (spikes disappear). Solution: Ensure that connections are tight. Degas mobile phase, use backpressure regulator after flow cell (for instance 100 psi, check flow cell specifications). |
| Drainage spiking (Charged Aerosol Detection, CAD) | Check for leaks at the waste bottle and drain/vent assembly. Check for the presence of internal tubing within drain line. |
| Nebulizer (Charged Aerosol Detection) | Nebulizer chamber may need to be cleaned. Remove column. Flush your charged aerosol detector with 80/20 water:methanol at 2.0 mL/min for several hours. Adjust flow-rate to 0.2–0.4 mL/min overnight. Re-check noise level of original mobile phase composition. |
| Column temperature significantly above boiling point of the mobile phase | Use backpressure regulator after the flow cell (for instance 100 psi, check flow cell specifications). Use a post-column cooler if available. Consider switching off the flow cell heating (FLD). |
| Grounding (Charged Aerosol Detection) | Ensure that the Charged Aerosol Detector is operating on the same circuit as the LC system and is plugged into a surge protector. |
| Column stored without caps | Flush column with strong mobile phase and make sure to store column properly, typically (RP-phases) in buffer-free, high organic liquid. |
| Electrical interferences | Make sure to exclude the previous causes of spikes first. Spikes are typically not random, but for instance related to cyclic strong power consuming equipment. In addition, spikes can simply be caused by current fluctuations in the power grid. Solution: Isolate the electrical circuit from strong current consumers or use an UPS (Uninterruptible Power Supply) to filter current fluctuations. |
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