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Troubleshooting
Tailing peaks, altered retention times, pressure fluctuations or missing detector signal - these and many more are all cases that are undesirable in the everyday life of the analyst, but nevertheless occur from time to time.
In order to help you with troubleshooting, some manufacturers have produced very helpful brochures on the subject of troubleshooting, which give you a good overview of possible sources of error and how to rectify them in everyday HPLC work. Below you will find possible causes and solutions for the most common problems.
We are happy to answer any questions and provide support on the subject of troubleshooting.
Technical Data
Peak tailing
Peak tailing is one of the most common problems in HPLC. The problem can have various causes. It is important to distinguish whether the problem affects all peaks or only individual peaks. If all peaks are affected, this indicates a physical problem in the system or column. This could be dead volume, a blocked frit or similar. If only individual peaks are affected, this indicates chemical problems, such as the tailing of bases on older columns.
| Observation | Possible cause | Solution/Prevention |
|---|---|---|
| Peak tailing with bases | Undesirable interaction of bases with active silanols | 1. use a more modern phase to reduce active silanols 2. add basic additives to the mobile phase to mask silanols (not necessary with modern phases) |
| Peak tailing of analytes that can form chelate complexes with metals | Unwanted interaction of analytes with metals in the stationary phase | 1. use a modern phase to reduce metals |
| Peak tailing with ionic/ionisable analytes | Unsuitable pH value in the mobile phase leads to undesirable interactions with charged silanols | 1. Check pka values of analytes and select pH value so that the analyte is fully charged or neutral 2. Use a pH value of 2.5 so that the silanols are uncharged to prevent ionic interactions |
| Peak tailing for all peaks | Blocked frit | 1. reverse column rinse (please observe manufacturer's information beforehand) 2. use inline filter |
| Peak tailing at all peaks | Void formation in the column | Reverse rinse 1st column (please observe manufacturer's information beforehand) Replace 2nd column |
| Peak tailing at all peaks | Dead volume in the system | 1. check all connections (fittings, correct cutting of capillaries) 2. reduce the number of connections 3. use shorter capillaries |
Peak Fronting
In addition to peak tailing, fronting is the second deviation from the ideal Gaussian shape of a peak. However, fronting does not occur as frequently as tailing. The most common reasons for fronting are injection volumes that are too large and an unsuitable solvent for the injection.
| Observation | Possible cause | Solution/Prevention |
|---|---|---|
| Peak fronting for all peaks | Injection volume or concentration too high, meaning that not every analyte can interact with the stationary phase and is eluted more quickly | 1. reduce injection volume 2. reduce the concentration of the analyte |
| Peak fronting for all peaks | Injection solvent is not compatible with the mobile phase (too strong or immiscible) | 1. use mobile phase as injection solvent 2. use injection solvent that is weaker than the mobile phase |
| Peak fronting for all peaks | Channel formation in the column due to improper handling (pH too high, pressure too high) | 1. observe the recommended pH and pressure range of the column 2. exchange column and replace with a column that is compatible with the desired pH and pressure range |
Peak splitting
Peak splitting can look different. If the peaks do not split very strongly, the peak splitting can appear as a broad peak. A shoulder of peaks is also possible and if the problem is very pronounced, two peaks may even be visible. The reason for peak splitting is usually a dead volume in the system or a partially blocked frit in the column and/or precolumn.
| Observation | Possible cause | Solution/Prevention |
|---|---|---|
| Peak splitting for all peaks | Partially blocked frit in the column | 1. reverse column rinse (please observe manufacturer's information beforehand) 2. use inline filter 3. if possible: replace the inlet frit of the column (please observe the manufacturer's information beforehand) |
| Peak splitting for all peaks | Partially blocked frit in the pre-column | 1. replace pre-column 2. use inline filter |
| Peak splitting for all peaks | Channel formation in the column due to improper handling (pH too high, pressure too high) | 1. observe the recommended pH and pressure range of the column 2. exchange column and replace with a column that is compatible with the desired pH and pressure range |
| Peak splitting stronger with early eluting peaks | Injection solvent is not compatible with the mobile phase (too strong or immiscible) | 1. use mobile phase as injection solvent 2. use injection solvent that is weaker than the mobile phase |
| Peak splitting with a peak | Coelution of an impurity | 1. improve sample preparation |
| Peak splitting at one peak | Coelution of a second peak | 1. optimise method to improve resolution |
| Peak splitting with ionisable compounds (acid/base) | pH value too close to the pka value of the analytes or mobile phase not sufficiently buffered | 1. select pH value 2 units away from pka value 2. increase buffer concentration |
Additional peaks
The problem with additional or unexpected peaks can have various causes. These peaks are often late eluting analytes from a previous sample or so-called memory effects from the injection process. However, so-called ghost peaks can also be the cause of additional peaks or impurities in the sample. So-called spikes are another problem. These are not "broad" peaks but short spikes.
| Observation | Possible cause | Solution/Prevention |
|---|---|---|
| One or more additional peaks | Impurities in the sample | 1. optimise sample preparation process |
| One or more additional peaks | Impurities in the mobile phase | 1. use HPLC grade solvent 2. use Ghost Guard columns |
| One or more additional peaks | Contamination in the column/pre-column | 1. rinse column 2. replace the precolumn 3. in case of frequent occurrence: Optimise method/introduce rinsing step |
| One additional peak | Late eluting peak from previous sample | 1. optimise method for the sample 2. rinse column after the run |
| Additional peaks comparable with other/previous sample | Memory effect from the injection process | 1. rinse injection system (sample loop, syringe) 2. optimise method parameters for the injection |
| Spikes | Air in the mobile phase or in the system | 1. degas mobile phases 2. check screw connections for leaks 3. if necessary, install a counter-pressure capillary on the detector (please note the maximum pressure of the detector cell) |
| Spikes | Air in the column (column not sealed with end plug) | 1. always store column with stopper 2. flush the column |
Negative peaks
Negative peaks are almost always due to the detector. The mobile phase shows a higher signal than the analyte, resulting in negative peaks during detection.
| Observation | Possible cause | Solution/Prevention |
|---|---|---|
| Negative peaks with the RI detector | Refractive index of analyte lower than that of the mobile phase | 1. use mobile phase with lower refractive index |
| Negative peaks in the UV detector | UV adsorption of the analytes is lower than that of the mobile phase | 1. change detection wavelength 2. use mobile phase with lower UV adsorption |
| All peaks negative | Signal polarity incorrectly set | 1. check polarity of the detector |
| All peaks negative | Detector cable connected incorrectly | 1. check wiring |
Missing peaks
Missing peaks can be another problem with peaks. The causes can be quite simple, such as the wrong solvent or no injection, but even a very strong tailing can make it appear as if no peak is eluting. Coelution is also conceivable if a peak is sensitive to a parameter such as pH or temperature.
| Observation | Possible cause | Solution/Prevention |
|---|---|---|
| No peak eluted | Incorrect solvent/mobile phase | 1. check solvent and mobile phase |
| No peak eluted | No injection has taken place | 1. check injection system (tightness) |
| No peak eluted | Wrong/no sample injected | 1. check whether the vial is full enough 2. check whether the correct vial was used |
| No peak eluted | Wrong column selected | 1. check the column used |
| No peak eluted | Incorrect flow rate set on the pump | 1. check the flow rate |
| No peak eluted | Leak in the system, causing the flow rate to decrease | 1. check connections |
| No peak eluted | Incorrect sample preparation | 1. check whether sample preparation was carried out correctly |
| No peak eluted | Detector set incorrectly | 1. check detector settings such as wavelength (UV) or mode (negative, positive for MS) |
| One peak does not elute | Matrix effects interfere with the sample | 1. optimise sample preparation 2. optimise separation method |
| One or more peaks do not elute | Adsorption in the vial (interaction with glass) | 1. use polymer vial or silanised vial |
| One or more peaks do not elute | Retention of analytes too strong | 1. optimise method 2. rinse column with strong solvent |
Retention time shift
If there is a shift in the retention time, it is important to know whether this shift occurs gradually or whether it occurred from one run to the next. This allows conclusions to be drawn about the cause. A constant shift, for example, indicates an ageing process of the column. If the shift occurs suddenly, this is more likely to be due to a problem in the system.
| Observation | Possible cause | Solution/Prevention |
|---|---|---|
| Retention time gradually decreases | Loss of the bound phase due to low pH value | 1. observe the recommended pH range of the column 2. exchange column or replace with column with different pH stability |
| Retention time gradually increases for polar/ionic compounds | Loss of the bound phase or endcapping allows increased interaction with silanols, often accompanied by increasing tailing | 1. observe the recommended pH range of the column 2. exchange column or replace with column with different pH stability |
| Retention time varies when using a new column | Differences in different lots of the stationary phase (can sometimes be quite large) | 1. check whether a column from a different lot was used 2. if deviations are too large, use original lot or adjust method |
| Retention has suddenly increased or decreased | Changes in the flow rate | 1. check whether the correct flow rate is set 2. check for leaks in the system 3. check for air bubbles in the system |
| Retention has suddenly decreased or increased | Column with a different dimension inserted | 1. check inserted column |
| Retention has suddenly decreased or increased | Column was overloaded and this leads to a disturbed peak shape, whereby the retention time also deviates | 1. check injection volume and concentration 2. check the column used (internal diameter) |
| Retention has suddenly decreased or increased | Incorrect composition of the mobile phase | 1. check the composition of the mobile phase 2. if a gradient valve is used, check all valves for functionality |
| Retention of an ionisable compound fluctuates greatly | pH value too close to the pka value of the analytes or mobile phase not sufficiently buffered, so that the smallest differences in pH value lead to large changes in retention | 1. select pH value 2 units away from the pka value 2. increase buffer concentration |
| Retention time varies from day to day or from morning to evening | Column temperature varies due to not tempering the column | 1. use column oven 2. operate column oven above room temperature to ensure stable retention in non-air-conditioned laboratories |
| Retention time of the new column differs from the old column | Column is not sufficiently equilibrated; this is particularly the case if TFA is used in the mobile phase | 1. equilibrate the column for longer (in the case of TFA also overnight) 2. if the storage time is short, store the column in the mobile phase with TFA |
Pressure too high
Excessive back pressure is a very common problem in HPLC and can have a variety of causes. It is important to differentiate whether excessive pressure occurs slowly or suddenly. A slow increase in pressure indicates normal ageing in the column/pre-column. If the pressure is too high, the column can be flushed or the precolumn replaced. As the pressure only increases slowly, this problem is often not immediately apparent. If there is a sudden increase in pressure, it is advisable to check whether maintenance work or something else has been carried out on the system beforehand. This may give an indication of the cause.
| Observation | Possible cause | Solution/Prevention |
|---|---|---|
| Pressure builds up gradually | Ageing of the column/column | 1. flush the column 2. if flushing does not show any improvement, replace the column/pre-column |
| Pressure is suddenly too high | Clogged frit | 1. flush the column in reverse (please observe the manufacturer's information beforehand) 2. use inline filter 3. if possible: replace the inlet frit of the column (please observe the manufacturer's information beforehand) |
| Pressure is suddenly too high | Clogged capillary | 1. systematically check capillaries |
| Pressure is suddenly too high | Clogged inline filter | 1. replace inline filter |
| Pressure is suddenly too high | Flow rate too high | 1. check the pump settings |
| Pressure is suddenly too high | Temperature too low, resulting in higher viscosity | 1. check the temperature of the column oven |
| Pressure is suddenly too high | Buffer/salt has failed | 1. check solubility of the additives used in all solvent compositions before using a new method 2. rinse the column |
| Pressure is suddenly too high | Wrong column (smaller inner diameter, longer or smaller particle size) | 1. check the column used |
| Pressure is suddenly too high | Incorrect capillaries used, can happen during maintenance/replacement of capillaries | 1. check capillaries used 2. document all maintenance/replacement work |
| Pressure is suddenly too high | Stainless steel fittings that are too tight can press in the capillary and create a bottleneck | 1. tighten fittings according to manufacturer's instructions 2. replace capillaries/connections |
| Pressure is suddenly too high | Incorrect solvent composition | 1. check the mixed mobile phase 2. with gradient valve, check valves for functionality |
Pressure too low
If the pressure is too low, this is almost always due to a leak in the system. If the pressure is too low, you should therefore always check for leaks first. In addition to leaks, flush valves can also be the problem if they have not been closed after flushing.
| Observation | Possible cause | Solution/Prevention |
|---|---|---|
| Pressure too low | Leak in the system | 1. search for leaks and repair connection 1. no obvious leak: check all connections |
| Pressure too low | Flush valve not closed | 1. close flush valve |
| Pressure too low | Pump non-return valve defective | 1. clean non-return valve (observe manufacturer's instructions) 2. replace non-return valve |
| Pressure too low | Flow rate too low | 1. check pump settings |
| Pressure too low | Temperature too high, resulting in lower viscosity | 1. check column oven |
| Pressure too low | No solvent in the supply so that the pump cannot deliver anything | 1. check solvent supply 2. flush air bubbles out of the pump if necessary |
| Pressure too low | Wrong solvent with lower viscosity used | 1. check solvent/mobile phase |
| Pressure too low | Incorrect solvent composition | 1. check mixed mobile phase 2. with gradient valve, check valves for functionality |
Baseline problems
Problems with the base line can be characterised in different ways. On the one hand there are randomly occurring problems and on the other hand cyclically occurring problems. Random problems are usually caused by impurities or air bubbles. This can even manifest itself as an additional peak in the form of spikes. Problems that occur cyclically or periodically indicate a problem with the detector, such as baseline drift or a temperature fluctuation.
| Observation | Possible cause | Solution/Prevention |
|---|---|---|
| Baseline drift | Detector not sufficiently stabilised | 1. flush detector with mobile phase until stable baseline is achieved (especially with gradient methods) |
| Baseline drift | Gradient method | 1. some detectors cannot be operated with a gradient (e.g. RI detector) 2. a certain drift can be normal with gradients |
| Baseline drift | Contamination in the detector cell | 1. flush the detector cell (follow the manufacturer's instructions) |
| Baseline drift | Temperature fluctuations during a run | 1. check temperature settings of the detector cell/column oven |
| Spikes in the baseline | Air bubbles in the system | 1. flush the system |
| Spikes in the baseline | Impurities in the column/mobile phase | 1. rinse column 2. prepare new mobile phase |
| Spikes in the baseline | Energy of the detector lamp no longer sufficient | 1. replace detector lamp |
| Strong noise in the baseline | Sensitivity of the detector set too high | 1. check the detector settings |
| Other baseline problems | Problem with the electronics of the detector | 1. contact manufacturer |
Downloads
ACE Troubleshooting HPLC
GL Sciences GC Troubleshooting Guide
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