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HPLC Troubleshooting

Mobile phase

The mobile phase composition significantly influences chromatographic performance and the resolution of compounds in the mixture being chromatographed. For accurate quantitative work, high-purity reagents and HPLC grade organic solvents should be used. The water used in mobile phase preparation, e.g. D.I. water and HPLC grade water, should have low conductivity and low UV absorption, appropriate to the intended use.

Solvent degassing is also a critical step for mobile phase  preparation. First degassing lowers the level of gas dissolved in the solvent. This process minimizes the chance of air bubbles getting into and becoming trapped in the detector cell. Second, degassing reduces the chromatographic effects due to air in the solvent. The most common means of solvent degassing
are ultrasonic agitation under vacuum for 10 to 15 minutes, vacuum filtration and sonication.

For gradient wash with detection wavelength below 210nm for related substances determination, extra care should be taken for mobile phase preparation. Follow method specified procedure to ensure all reagents and solvent are in required purity or higher level and all glassware are rinsed with appropriate solvent. Noisy baseline may be observed if solvents, reagents
or containers contain any interference impurities. If at all possible, the analysis wavelength should be at least 20 nm above the UV cutoff.

Exchange of immiscible solvents in column/system

Stop the pump flow, remove any column from the HPLC system and use a union to connect the inlet and exit tubing.

Change HPLC system from normal phase to reverse phase

Put about 200 mL of IPA (wash solvent) into the mobile phase reservoir. Carefully transfer the inlet filter assembly into the reservoir and start the pump with 0.5mL/min flow rate, slowly increase the flow rate to 2 mL/min, and pump about 5 system volumes of the solvent (or about 30 minutes at 2mL/min) through the system to wash out all of the previous mobile phase. Then put new mobile phase on the system (when buffer is used in the new mobile phase, the system should be flushed at 2 mL/min with 5 system volume of the same aqueous-organic mobile phase without buffer before converting to 100% new
mobile phase), wash out all of the wash solvent from the system with 5 system volumn at 2mL/min (or about 30 minutes at 2mL/min)

Change HPLC system from reverse phase to normal phase

Put about 200 mL of 50% MeOH (wash solvent) into the mobile phase reservoir. Carefully transfer the inlet filter assembly into the reservoir and start the pump with 0.5mL/min flow rate, slowly increase the flow rate to 2 mL/min, and pump about 5 system volumes of the solvent (or about 30 minutes at 2mL/min) through the system to wash out all of the previous mobile
phase. Stop the pump, and place the inlet filter assembly in 200mL of IPA. Flush the system at 2mL/min for 5 minutes. Then put new mobile phase on the system to wash out all of the wash solvent from the system with 5 system
volumes at 2mL/min (or about 30 minutes at 2mL/min)

Change mobile phase from buffer solution to pure or high concentration of organic solven

In order to avoid salt precipitation to occur, HPLC system/column should be flushed with DI water, followed by 50% MeOH for about 5 system volumes respectively. Then change the wash solvent to mobile phase with high concentration of organic solvent to condition the system until good baseline obtained (see section 5.1 for detail).

Example 1, from 100% organic solvent to the mobile phase that contains buffer

Assume that we are dealing with a reverse-phase column/system that was stored in acetonitrile, and that we want to use a mobile phase consisting of a 1:1 mixture of a buffer and methanol. In order to avoid a precipitation of the buffer in the column/system, we should first purge the column/system with a few column/system volumes (1-3 is enough or 50-100 mL) of
a 1:1 mixture of methanol and water before we switch to the methanol-buffer mobile phase. You should also be aware that the column backpressure will increase roughly 5-fold during this conversion, due to the viscosity difference
between acetonitrile and the methanol-water mixture.

Example 2, from hexane (normal phase) to aqueous buffer as mobile phase (reverse phase)

 Assume that we would like to apply an aqueous buffer as mobile phase on a column/system stored in hexane. This conversion needs to be planned carefully. Hexane is only partially miscible with methanol and acetonitrile, so we need another solvent between hexane and the polar solvent. Also on the aqueous side, we need to use water as an intermediate between the buffer and the polar solvent. Therefore, the complete conversion would require 1-3column/system volumes (or 50-100 mL) each of ethyl acetate (or IPA or acetone), methanol (or acetonitrile), water, and finally the buffer that was intended as mobile phase.


The dimension and packing materials of the column must be specified in the method. Columns should be reserved for one assay type and used exclusively for that purpose. A suitable guard column may be used whenever possible.

Before using a new column, check manufacturer’s instructions for any conditioning requirements.

The performance and lifetime of bonded-phase columns are best preserved by storing them in 100% organic solvent (preferably acetonitrile) where possible. Storage with buffered solutions (particularly those containing high concentration of water and alcohols) should be avoided. When buffers are used, columns should be flushed with 10 to 20 column volumes
of the same aqueous-organic mobile phase without buffer before converting to 100% organic for storage. Flushing densely
bonded C18 columns with pure water should be avoided. Columns should be capped tightly during storage to prevent the packed bed from drying out.

A wash method may be set up and placed at the end of the sequence. Use HPLC grade water or the appropriate solvent, in a separate pre-primed line to remove all buffer salts from the HPLC system. Approximately ten column
volumes of solvent are usually sufficient to wash out the buffer salt in the column. Where ion pair or amine modifier reagents have been used, prolonged washing may be required, especially when SDS is used as medium for dissolution
test. Never leave the system filled with 100% water, as bacteria or mould will grow inside the system. Specific column wash procedures and storage conditions may be described in the method.

System Suitability Failures

If standard and sample preparations are still within their expiration periods, the chemist may re-inject the samples and standards beginning at the point in the sequence where the problem occurred, ensuring that all system suitability, % Recovery of standards, % RSD and of agreement of bracket standards requirement are met.

Changes to validated chromatographic methods should not be made unless supported by robustness data.

Changes to compendial methods may be allowed using the guidelines described in USP <621>. If adjustments of operating conditions are necessary to meet system suitability requirements, each of the following is the maximum variation that can be considered.

pH of mobile phase: within ±0.2 unit of the value or range specified.

Concentration of salts in Buffer: within ± 10%, provided the permitted pH variation is met.

Ratio of components in mobile phase: To minor components, ± 30% relative. However, the change in any component can not exceed ± 10% absolute.

Column length: ± 70%.

Column inner diameter: ±25%.

Particle size: Can be reduced by as much as 50%.

Flow rate: ± 50%.

Injection volume: Can be reduced as far as is consistent with accepted precision and detection limits.

Column temperature: ±10°C.

Poor peak-shape

Column overload: only the large peaks are distorted, but small peaks give good peak shape, probably overload problem.

Column collapse: the same peak distortion throughout the chromatogram.

Extracolumn effects: Broad and tailing peaks are observed at low retention, but symmetric peaks and high plate count at large retention, then the cause of the problem is most likely some extracolumn effects due to sample dispersion which occurs in connecting tubing with big internal diameter, sample valves, flow cells and in column end-fittings with big void volume.

Basic analyte tailing: secondary interactions with silanols on the column.

Solvent incompatibility: Solubility limit of analytes in the mobile phase, sample solvent or components interfere with the adsorption of the sample on the column top.

Second equilibrium: The analyte itself exists in one or more forms or conformations that can be distinguished chromatographically and that is in equilibrium with each other when pH value or salt concentration of mobile
phase varies. For example, the analyte peak may broaden or become two separate peaks when the pH value of mobile phase is close or same as the pKa value of the analyte.

Detector overload: Flat top peak

Retention time changes

The final retention time of a peak is uniquely determined by following variables:

The structure of the analyte; the column (The physical characteristics and surface chemistry of the packing materials; the dimension of the column); the composition of mobile phase; the temperature of the column; mobile phase flow rate and the dead volume in the system; the composition of the dilution solvent containing the analyte; the volume of the injection.

Retention time drift

Possible cause: Poor temperature control (solution: Thermostat column); Mobile phase changing (Prevent mobile phase evaporation, reaction, etc.);

Poor column equilibration (Allow more time for column equilibration between runs, especially ion-pair reagents used in the mobile phase); pH change (Remove mobile phase from the system and re-adjust pH of mobile phase).

Abrupt retention time changes (compared with previous injection or previous run)

Possible cause: Flow rate change (Reset flow rate)

Air bubble in pump (remove air from pump by purging)

Improper mobile phase (a. Replace with proper mobile phase,
b. Set proper mobile phase mixture on controller).

Improper column (change a proper column)

Problem with the HPLC system, i.e. pump problem (change a


If at all possible, the analysis wavelength should be at least 20 nm above the UV cutoff of mobile phase or use mobile phase components that have cut of at least 20nm below desired monitoring wavelength.

To avoid the use of solvent systems that generates detector response, acetonitrile and phosphate buffer are predominantly used in gradient HPLC-UV work.

Noisy baseline:

Detection wavelength is too near the UV cutoff.

System not fully equilibrated, continue to flush with mobile phase, particularly dirty sample injected.

Gradient wash, one or more of the mobile phase components is contaminated.

Dirty detector, clean the cell or low detector lamp energy.

Sensitivity setting has been lowered, baseline appears noisier.

Air trapped in HPLC system, pump, column and detector cell, etc. (Note: if any air bubble is trapped in the pump head, then a big pressure fluctuation may be observed, i.e. pressure fluctuation is more than 5 bars. Purge the pump head at a high flow rate, e.g. 10mL/min, to remove the air bubble in the pump head. If a noisy baseline is observed, probably some air
bubbles are trapped in the detector cell or elsewhere in system, purge the system with mobile phase or with 100% methanol or IPA).

Sudden increased system backpressure

Particles in dirty sample block the system.

The introduction of immiscible solvents generates high viscosity of solvents.

Insoluble materials participate in the system.

Loss of efficiency and resolution

Column aging, buildup of nondetectable contaminants on the column.

Improper preparation of the mobile phase or change in the system temperature.

Improper nuts/ferrules unions.

Changes in sample matrix or sample preparation.

Unanticipated peaks (extra peak or ghost peak) in the chromatogram

Change in samples, contaminants in samples, carryover and contaminants in mobile phase that is for gradient wash.

Erratic results

Check integration parameters, sample and standard preparation, leaks/air bubbles in system, injector to make sure injection precision and correct injector wash, detector source output energy, sample in correct solvent and the data acquisition parameters.


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